Entitled Expression of small RNA correcting method and apparatus

Technical field

[0001]

　The present invention relates to a device for correcting the methods and the expression level for correcting the expression level for comparative analysis of the expression level of a target small RNA contained in a plurality of analytes.

Background technique

[0002]

　non-coding and the RNA (ncRNA), it is a general term for RNA that does not code for a protein is classified roughly into a regulatory system of the RNA housekeeping RNA. There are ncRNA of various lengths, molecular especially less than 200 bases is called a small RNA (small RNA).

[0003]

　The housekeeping RNA, ribosomal RNA (rRNA), transport RNA (tRNA), small nuclear RNA involved in splicing (snRNA), Small nucleolar RNA involved in rRNA modification (snoRNA) or the like is known ing.

[0004]

　For regulatory systems RNA as play an important function factor to elucidate the biological functions, which in recent years in particular attention, an important role in the regulation and gene silencing mechanism intracellular distribution of gene expression and RNA that it is responsible for are being revealed recently. Gene silencing mechanism that this regulatory system RNA functions is referred to as RNA interference (RNAi), is revealed in experiments using nematodes in 1988, then, obviously the presence of similar mechanisms in Drosophila and mammalian cells It became. ncRNA as the regulatory system RNA is chain length of approximately 20-25 bases, their mechanism of action, and translational repression by micro RNA (miRNA), cut and target DNA of the target mRNA by small interference RNA (siRNA) It is classified roughly into a gene silencing via heterochromatinization region.

[0005]

　miRNA is, genomic DNA Caracalla hairpin-like structure RNA (precursor) come transcribed as. This precursor, dsRNA cleavage enzyme (Drosha, Dicer) having a specific enzyme RNase III cleavage activity after being cut by, changes to the form of double-stranded, then a single strand. And, incorporated into a protein complex antisense strand of one is referred to as a RISC, are thought to be involved in mRNA translation inhibition. Thus, miRNA after transfer, since the embodiment differ in each stage, usually, when the miRNA target (detection target) is hairpin structure, a double-stranded structure, such as a single chain structure it is necessary to take into account the various forms. miRNA comprises 15 to 25 bases of RNA, and its presence is confirmed in a variety of organisms.

[0006]

　Recently, miRNA not only the cell, various serum is samples without cells (biological samples), blood plasma, urine, abundantly present in the body fluid of the spinal fluid or the like, that its expression level, including cancer likely to be biomarkers of disease have been suggested such. miRNA June 2014 There are currently more than 2500 species in humans (miRBase Release 20), when using a measurement system such as a high sensitivity DNA microarrays, expressed miRNA over 1000 species of which in serum and plasma it is possible to detect simultaneously. Therefore, the serum or plasma using the DNA microarray method, urine, biomarker discovery study of body fluid of spinal fluid and the like are performed.

[0007]

　When performing gene expression analysis using a DNA microarray, the sample and experimenter, the experimental conditions, it is well known that errors in the resulting measurement (data) occurs. Therefore, the correction method for measurement for correcting the errors have been devised. The correction, be any sample, the measurement of the expression amount of a plurality of genes and lump, which taken as a gene expression data set, the method premised on the principle that there is no difference in the expression level is usually well-used is, global normalization method, quantile method, lowess method, or the like 75 percentile method. However, these correction methods, there is a drawback that can be used only when it detects the exhaustive gene certain number or more in.

[0008]

　On the other hand, paying attention to a particular gene expression level is identical between the specimens (such as beta-actin or GAPDH), as measurement of that gene is constant, has been performed a method for correcting the data for each sample .

[0009]

　Even when analyzing small RNA by DNA microarray, a global normalization method used in gene expression analysis as described above, quantile method, lowess method, 75 percentile method such correction method but is used, only detect a particular gene If you do not these methods can not be used. As a method other hand expression of a particular gene is corrected to be constant, of the small RNA expressed in the sample, the housekeeping RNA (U1snoRNA, U2snoRNA, U3snoRNA, U4snoRNA, U5snoRNA, U6snoRNA, 5SrRNA, 5.8 method of correcting using SrRNA) has been proposed (Patent documents 1 and 2).

[0010]

　Patent Document 1, Patent Document 2, when detecting the miRNA are small RNA, as the detected value of 5SrRNA detected simultaneously is constant in all the samples, but to correct the detection result of the miRNA, these no assurance that a certain amount expressed between sample

[0011]

　In Patent Document 3, when detecting the miRNA are small RNA, to detect the mRNA simultaneously, but to correct the detection result of the miRNA in the representative value, the normal distribution of the mRNA this method was also detected is guaranteed Ruaru a method that can be applied to the case of detecting a predetermined number or more of the number of mRNA.

[0012]

　Therefore, in order to correct the errors of gene expression between experiments, the standard is a nucleic acid used in the experiments step, it is proposed to correct the error between experiments using the abundance that the detected and that (Patent documents 4-6). Patent Document 4 and 5, the arrangement and method for designing a probe nucleic acid for detecting the reference material or the reference material is a nucleic acid present, and accuracy is shown in the amplification step and detection step, detection methods These although it is possible to evaluate the performance, it is not shown to actually perform error correction of between experiments involving extraction process of nucleic acids from the sample. Although shows an arrangement of the standard substance is a nucleic acid for correcting the error of the detected value of the gene expression in even sample Patent Document 6, and using this sequence from the amplifying of nucleic acid, the amplification step We can only correct an error between experiments.

[0013]

　That is, the method shown in Patent Documents 4-6 described above, there nucleic acid extracted from the sample is a sufficient amount, only if the nucleic acid to be used can be precisely quantified, measured including amplification and detection of nucleic acids steps of it can be evaluated and an error correction accuracy when measuring result of the correction between the actual experiment, when dealing with body fluids as if or samples, particularly dealing with sample traces, the target small RNA extracted amount becomes very small amount, because the small RNA amount can not be accurately measured, it is impossible to perform substantially corrected by this method. Therefore, not only the step of detecting small RNA, is possible to correct the error between experimental extraction step was also included from the sample is very important.

[0014]

　Therefore, as a method of evaluating and correcting an error between experiments, including the extraction step from the specimen, a method of using the standards it has been considered so far, is the same base length of a nucleic acid and a small RNA It is corrected by using the standard materials have been studied. For example, similar to the miRNA as shown in Non-Patent Document 1 20 bases about short RNA is base length was used as a standard substance, which was subjected to extraction with a certain amount put into the specimen, of each experiment how to correct an error of the extraction process of the target small RNA in the extraction process has been proposed.

CITATION

Patent Document

[0015]

Patent Document 1: Laid-Open Patent Publication No. 2007-75095
Patent Document 2: Laid-Open Patent Publication No. 2007-97429
Patent Document 3: Laid-Open Patent Publication No. 2014-007995
Patent Document 4: Laid-Open Patent Publication No. 2011-239708
Patent Document 5: Patent First Bulletin No. 5,229,895
Patent Document 6: the United States is willing to open a charter No. 2010/0184608 book details

Non-patent literature

[0016]

非特許文献1 : Nobuyuki Kosaka Edit.,「Circulating MicroRNAs : Methods and Protocols （Methods in Molecular Biology）」, p1-p10, Human Press, New York（2013）

Summary of the Invention

Problems that the Invention is to Solve

[0017]

　When comparing analyzing the expression level of the target small RNA in between specimens, it is necessary to correct the difference between the extraction efficiency in the step of extracting the nucleic error experimental conditions between analyte, in particular sample. As a method has been often used so far, there is a method of using a global normalization and housekeeping RNA, for the above as small RNA, it is necessary to detect exhaustive number of small RNA, constant between sample There are such disadvantages that housekeeping RNA ensuring expression amount is not present, it can not be said to be effective in comparative analysis.

[0018]

　As for the correction method using the standard substance, as described above, by performing the correction using the standard is a nucleic acid of the same base length and small RNA targeting has been investigated, actually these with small RNA similar base long RNA as a standard, in particular when using body fluids as a sample, the influence of various contaminants contained therein and various conditions of the sample, the extraction efficiency of the standard material from the specimen It is not stable, the result measured value is not stable as, for accuracy can not be guaranteed, could not be used to correct the measurement results between experiments.

[0019]

　As described above, in the past, for comparative analysis of the expression level of a target small RNA extracted from each sample, it is possible to accurately correct the measured value of expression amount of between specimens, utilizing standard effective correction method that did not.

Means for Solving the Problems

[0020]

　Inventors intensively studied, in the expression amount of the correction method for comparative analysis of the expression level of a target small RNA contained in a plurality of analytes, nucleic acid length than small RNA for a plurality of analytes fixed amount added as standard substances is considerably longer 200 or more bases of a nucleic acid in the sample, was extracted from the sample nucleic acid, was measured in the presence of standard with the expression level of each target small RNA extracted, the presence of a standard by performing correction using the measurement of the amount, more accurately conventionally found that it is possible to perform the expression amount of the correction between each sample, and completed the following invention.

[0021]

(1) a plurality of correction of the expression level for comparative analysis of the expression level of a target small RNA in a specimen method,
　the plurality of each sample, at least one standard nucleic acid length is 200 bases or more nucleic acid after addition of substances, extraction step extracts nucleic acid from the sample to obtain a nucleic acid sample;
　the amount of target small RNA and standards present in each nucleic acid sample extracted was measured respectively, the target small RNA for each sample obtain the expression levels and measurements of the amount of extracted standard, measuring step;
　for each sample, to obtain a representative value from the measured value of the amount of extracted standard, the representative value acquiring step;
　optionally terms extracted amount of standard a set reference value, the Samata the ratio of representative values of the standard of each sample obtained by the representative value acquiring step acquires as the correction coefficient of the target small RNA expression levels for each sample, the correction coefficient acquiring step;
　their acquired each correction coefficient was ; Correction step of respectively using, for correcting the expression level of the measured target small RNA for each sample
containing a correction method.
(2) Correction method according to the nucleic acid length of the standard substance is 200 or more bases 1200 bases or less (1).
(3) at least one standard comprises at least one selected from standard is a nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1-5 and 15-17, the correction method described in (2).
(4) use of two or more kinds of standards, the correction method according to any one of (1) to (3).
(5) Correction method according to any one of from the specimen is a body fluid derived from the specimen (1) to (4).
(6) Correction method according to any one of from the target small RNA is miRNA (1) to (5).
(7) Extraction of nucleic acid sample in said extraction step is carried out by phenol-chloroform method, the correction method according to any one of (1) to (6).
(8) the measurement step, a labeled nucleic acid sample with a labeling substance, a probe for capturing a plurality of target small RNA immobilized on a support and at least one probe for trapping standards by contacting performs hybridization and correction method according to any one of comprising obtaining an extract of the expression level and standard of each target small RNA as signal strength measurements, (1) to (7) .
Representative value obtained by (9) the representative value acquiring step, was calculated from the measured value of at least one standard extraction amount, the average or median, expressed in logarithmic value, (1) to correction method according to any one of (8).
(10) the reference value, a fixed numerical value which is arbitrarily set with respect to the extracted amount of standards, or, in the representative value of the standard substance extracted amount obtained for the first analyte which is arbitrarily selected from the plurality of samples there, the correction method according to any one of (1) to (9).
In (11) the correction step,
in (a) the correction coefficient acquisition step, when obtaining a value obtained by subtracting the reference value from the representative value as the correction factor, the correction factor from the measured value of expression amount of the target small RNA subtracting,
(b) in the correction coefficient acquiring step, to acquire the value obtained by subtracting the representative value from the reference value as a correction factor, adding the correction factor to the measured value of expression amount of the target small RNA,
in (c) the correction coefficient acquisition step, when obtaining a value obtained by dividing the representative value with the reference value as a correction factor, is divided by the correction factor the measured value of expression amount of the target small RNA, or
(d) in the correction coefficient acquiring step, to acquire the value obtained by dividing the reference value in the representative value as the correction coefficient, multiplying the correction coefficient to the measured value of expression amount of the target small RNA,
by performing the correction, ( 1) to (10) The method according to any one of.
(12) An apparatus for correcting the expression level to compare analyzing the expression level of a target small RNA in a plurality of specimens,
　in each of a plurality of analytes, at least one standard nucleic acid length is 200 bases or more nucleic acid after addition of materials was measured using a nucleic acid sample obtained by extracting the nucleic acid from the sample, storage means for storing the measured values of the target small RNA expression levels and standards extraction amount for each sample ;
　for each sample, to obtain a representative value from the measured values of standard extraction amount, and the representative value acquiring means;
　and optionally set reference value with respect to the amount of extracted standard, each obtained by the representative value acquiring means the Samata the ratio of the value representative of the analyte, respectively acquires a correction coefficient of the target small RNA expression levels for each sample, the correction coefficient acquiring means and;
　using each correction coefficient acquired by the correction coefficient acquiring section , the each Correcting the expression level of the measured target small RNA in the body, a correction unit
including said device.
(13) the representative value is calculated from the measured value of at least one standard extraction amount, an average value or median was represented by logarithm, according to (12).
(14) according to the target small RNA is miRNA (12) or (13).
(15) said correction means,
in (a) the correction coefficient acquiring section, when acquiring a value obtained by subtracting the reference value from the representative value as the correction factor, the correction factor from the measured value of expression amount of the target small RNA subtracting,
(b) in the correction coefficient acquiring section, when acquiring a value obtained by subtracting the representative value from the reference value as a correction factor, adding the correction factor to the measured value of expression amount of the target small RNA,
in (c) the correction coefficient acquiring section, when acquiring a value obtained by dividing the representative value with the reference value as a correction factor, is divided by the correction factor the measured value of expression amount of the target small RNA, or
(d) in the correction coefficient acquiring section, when acquiring a value obtained by dividing the reference value in the representative value as the correction coefficient, multiplying the correction coefficient to the measurement value of the expression amount of the target small RNA,
it performs the correction by, ( 12) to (14) Apparatus according to any one.
(16) wherein are stored in the storage means, the measured value of expression amount and standards extraction amount of the target small RNA in a plurality of samples, a plurality of labeled nucleic acid sample with a labeling substance, immobilized on a support probe and at least one standard for capturing a target small RNA are contacted with the probes to capture performs hybridization, expression level and the signal strength measurements extraction quantity of the standard substance of each target small RNA is a value measured respectively as a device according to any one of (12) to (15).
(17) in order to correct the expression level for comparative analysis of the expression level of a target small RNA among a plurality of analytes, one or more computers,
　in each of a plurality of analytes, nucleic acid length 200 bases or more nucleic acid after addition of at least one reference material is, by using a nucleic acid sample obtained by extracting the nucleic acid from the sample, the amount of target small RNA and standards present in each nucleic acid sample were measured to obtain a measure of the expression level of the target small RNA and the extracted amount of standard for each analyte, the measuring step;
　for each sample, to obtain a representative value from the measured value of the amount of extracted standard, the representative value acquiring step;
　standard a reference value which is arbitrarily set with respect to the amount of extracted substances, the correction coefficient of the target small RNA expression levels for Samata is a specific, each sample of a representative value of the standard of each sample obtained by the representative value acquiring step It is obtained as the correction coefficient acquiring step And
　the obtained respective correction coefficients were used respectively, the correction step of correcting the expression level of the measured target small RNA for each sample
program for executing.
(18) in order to correct the expression level for comparative analysis of the expression level of a target small RNA among a plurality of analytes, one or more computers,
　in each of a plurality of analytes, nucleic acid length 200 bases or more nucleic acid after addition of at least one reference material is, was measured using a nucleic acid sample obtained by extracting the nucleic acid from the sample, the measurement of the target small RNA expression levels and standards extraction amount for each sample ; storage means for storing a value
　for each sample, to obtain a representative value from the measured values of standard extraction amount, the representative value acquiring means;
　and a reference value which is arbitrarily set with respect to the amount of extracted standard, the representative value acquiring means the Samata the ratio of the representative value of each sample obtained by, respectively acquires a correction coefficient of the target small RNA expression levels for each sample, the correction coefficient acquiring means; and
　each obtained by the correction coefficient acquiring section correction Using equation corrects the expression level of the measured target small RNA in each sample, the correction means
programmed to function as a.
(19) (17) or (18) recording a program described in a computer-readable recording medium.
(20) and probe for capturing a plurality of target small RNA, SEQ ID NO: 1 to nucleotide sequence of 5 and 15-17 for capturing at least one standard selected from standard is a nucleic acid comprising a support and the probe is immobilized, small RNA expression analysis chip.

Effect of the Invention

[0022]

　According to the present invention, measuring the expression level of small RNA extracted from the specimen, when comparing a small RNA expression levels between the specimen can be conventionally corrected accurately target small RNA expression levels. Thus, comparative analysis of the target small RNA between the analyte will be able to practice more accurately.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

FIG. 1 is a conceptual diagram of a method of the present invention.
Is a block diagram schematically showing the configuration of the analysis apparatus of the present invention; FIG.
[3] is an example of a flowchart of a correction process of the target small RNA expression levels according to the present invention.
[FIG. 4] (A) is a scatter plot after correction was embodiments correct the scatter plot after performing the 2, and (B) Comparative Example 2.

DESCRIPTION OF THE INVENTION

[0024]

　Standards referred to in the present invention, in the measuring step from the extraction step, is stably coexist in the sample containing the target small RNA, to perform the determination of the presence of the reference material together with the measurement of the expression amount of small RNA of interest Accordingly, a substance used to obtain a reference for correcting the variation of the measured value of expression amount of the target small RNA between the measurement of multiple analytes (variation or error between measurements). That is, based on the abundance of standards, it is possible to correct the measured value of expression amount of the target small RNA of between measurements of multiple analytes.

[0025]

　First, the concept of a correction method for the expression of a target small RNA using the present invention standard will be described with reference to FIG.

[0026]

　In Figure 1, labeled nucleic acid extracted from a sample, the probe for capturing a plurality of types of target small RNA (hereinafter also referred to as "small RNA capture probe".) And probe for capturing the standard (hereinafter, also referred to as "standard capture probe".) is the result of detection by the fixed microarray schematically shows the histograms of the signal values. Hereinafter, a probe for capturing the probe or reference material for capturing small RNA, also referred to collectively "capture probe" or simply "probe".

[0027]

　In Figure 1A, the results of the target small RNA extracted from the specimen A and the specimen B, respectively and analyzed using DNA microarrays are indicated by the histogram. Distribution of measurements obtained from a plurality of target small RNA capture probe mounted on the microarray (histogram), and a plurality of the representative values ​​of the measurements obtained from the standard capture probe is shown, respectively. In the sample A and sample B, histogram of small RNA greatly deviate. Therefore, it can be interpreted that there is a large difference in the expression level of small RNA between samples. On the other hand, experimental error, can be interpreted also as the difference by the nucleic acid extraction efficiency occurs particularly in the nucleic acid extraction process from the analyte. Which is correct, it is not possible to determine from the histogram only.

[0028]

　Shown in FIG. 1A, a representative value of the measurement values ​​obtained from the standard capture probe is a nucleic acid shows almost the same value in sample A and sample B. That is, sample A and sample B is being subjected to correctly experiment, it can be determined that the experimental error no. In this case, it would be that there is a large difference in the expression level of small RNA between analyte AB, the correction of the measured values ​​of the small RNA when the comparisons between the analyte is not required.

[0029]

　1B, the are the results of analysis of the specimen C and specimen D using a DNA microarray is schematically shown. It shows a histogram of the measurements obtained from a small RNA capture probe, and a representative value of the measurement values ​​obtained from the standard capture probe, respectively.

[0030]

　In the sample C and the sample D, a histogram of the measured values ​​of the small RNA indicates a similar distribution. On the other hand, the representative value of the measurement values ​​obtained from the standard capture probe is significantly deviated in sample C and sample D. The detection result of the sample C and the sample D This indicates that experimental error is caused by some reason. In such a case, upon the comparison between the sample CD, it is necessary to appropriately correct the measured values ​​of the small RNA.

[0031]

　In accordance with the present invention, showing a histogram after correction of the measured value of the target small RNA in Figure 1C. Specific methods of correction is described as follows. As the measurements obtained from the standard capture probe sample C and sample D match, data were corrected for sample C. By this correction, the representative value of the measurement values ​​obtained from the standard capture probe is as a matching specimen C and specimen D, the histogram of the measured values ​​of the corrected target small RNA capture probe using the same correction factor , come largely shifted. In other words, even between specimens CD, it comes to a large difference is the expression amount of small RNA.

[0032]

　In the present invention, comparative analysis of the expression level of a target small RNA among a plurality of specimens (measurement) to. Specimen number may be two, or may be three or more. The measurements across multiple analytes here include different measurement of a plurality of types of target small RNA, measurement of each sample when measuring a plurality of times the same target small RNA, or measurement that combines both .

[0033]

　The "small RNA" in the present invention, base length made in vivo means of less than 200 base RNA. For example, ribosomal RNA (5S rRNA, 5.8S rRNA), transfer RNA (tRNA), small nuclear ribonucleoprotein particle RNA (snoRNA), small nuclear RNA (snRNA), or a micro RNA (miRNA), immature before undergoing processing as miRNA, but like miRNA / miRNA duplex stem loop pre-miRNA or duplex can be exemplified, but not limited to. Examples of preferred small RNA, can be given miRNA.

[0034]

　In the present invention, in the extraction step and measurement step for comparative analysis of the expression level of a target small RNA, there reference material at a constant amount to the target small RNA. In particular, in the extraction step, standard is a nucleic acid is preferred to be extracted by the same extraction efficiency and the target small RNA.

[0035]

　Standards for use in the present invention is a nucleic acid. The nucleic acid length is a long 200 bases or more than small RNA targeting, preferably 200 bases or more 1200 bases or less, more preferably 500 bases or more 1200 bases or less. Generally single-stranded RNA, as long as the nucleic acid length of 200 to 300 bases or more, it becomes easy to form hydrogen bonds in the chain, physically or stabilized, various salts and lipids, proteins, etc. and by or associated, it can be kept more stable state even chemically. On the other hand, if it nucleic length of the standard substance is less than 200 bases is the nucleic acid, increased variability among experimental conditions and sample conditions during extraction efficiency and the measurement result is extracted from the sample, due to the influence of contaminants contained in the sample , it is feared to be especially extracted by different extraction efficiency and small RNA.

[0036]

　Also, standard is less (1), preferably have the properties of (2).
(1) GC content: in the range of 30-70%.
(2) Tm value is 10 ° C. or higher 95 ° C. or less.

[0037]

　GC content: of these, (1), A in the nucleotide sequence of the reference material to be used, T, G, C G in all bases can be determined from the presence ratio of C. Since GC content: increase in the number of higher hydrogen bond, the structure of nucleic acids, the nature tends to stabilize, the sequence specificity is decreased when too high a measurement. Therefore, it is preferable that GC content: reference materials is in the range of 30-70%, and more preferably in the range of 40-60%.

[0038]

　Tm value (2), based on the nucleotide sequence of the reference material, nearest base pairs (Nearest Neighbor) method (PNAS, 1998, 95: 1460-1465), etc. can be calculated by using the. In general, it is said to have high structural stability of a nucleic acid as Tm value is higher, the Tm value of the standard substance is preferably at 10 ° C. or higher 95 ° C. or less, more preferably 30 ° C. or higher 95 ° C. or less, it is further preferred 86 ° C. or more and 95 ° C. or less.

[0039]

　Standards for use in the present invention, considering the measurement step, it is preferable to select those which do not cross-hybridize with genetic transcripts contained in the sample to be used. Selection Specifically, for any gene transcripts of the same species as the target small RNA that has been recorded in public databases, sequence homology the nucleic acid is 50% or less, utilizing the homology search program can do. Here, the homology search program is not particularly limited, for example FASTA, can be applied BLAST, public programs like Mega Blast. As the public databases, but are not limited to, Genbank sequence information of the gene transcripts are stored (NCBI), EMBL (EBI), can be used Ensembl, a database such as miRBase.

[0040]

　Standard used in the present invention, by applying the organic chemical synthesis of nucleic acids, or a method of synthesizing in a host microorganism such as E. coli an array of standard using vector incorporating such a plasmid, standard incorporate RNA synthetase can recognize sequence such as T7 promoter upstream of the sequence of the substances, by applying the biological synthesis methods such as a method of synthesis by an enzyme such as T7 polymerase can be prepared. Further, nucleic acid calibrator that can be used as a reference for validity evaluation and quality control of the analyzer and analysis method are known, since there commercial products as standards in the present invention such known ones it can be used.

[0041]

　The standard not only those of the state of the single strand may also include those of the duplex formed with a complementary strand. Further, the purpose is to match the small RNA and chemical properties to be targeted, be included in a part of the nucleotide sequence of naturally occurring nucleic acids, it may comprise a nucleotide sequence not naturally occurring. Further, the same base sequence may be a sequence which is disposed a plurality of times in a plurality of times repeated or random, may contain an initiation codon and termination codon to a part of the sequence. Further, on both sides or one side of the sequence primers sites such as poly A may be granted.

[0042]

　Standard is preferably a DNA or RNA, may be used PNA, nucleic acid derivatives such as artificial nucleic acids such as LNA. Here, the nucleic acid derivative, labeled derivatives due fluorophore, modified nucleotides (e.g., halogen, alkyl such as methyl, alkoxy such as methoxy, thio, nucleotides include groups such as carboxymethyl, and reconstruction of a base, double bond saturation, means deamination, the derivative comprising a nucleotide, etc.) that received such substitution of sulfur molecules oxygen molecules. Further, end may be modified with various functional groups, phosphoric acid group or an amino group and a thiol group as such functional groups.

[0043]

　In the method of the present invention, to standards may be used one type may be used in combination. It may also be used in a state of being encapsulated in vesicles formed by proteins and associated state or lipids.

[0044]

　As the specimen that may be used in the methods of the present invention is not particularly limited, for example, blood, serum, plasma, urine, feces, spinal fluid, saliva, swabs, cerebrospinal fluid, sweat, tears, semen, lymphatic fluid, various tissue fluid or body fluid, such as synovial fluid, various tissues, frozen specimen and paraffin-embedded specimens (FFPE) or sections thereof of the cell culture solution cultured cells and tissues, such as the sample separated from a living body, namely biological samples and the like. Further, it is possible to include various food or dilution thereof or the like. Since used by adding a certain amount of standard constant amount of the specimen, it is preferable that particularly a body fluid. Further, a plurality of analyte comparative analysis is different organizations may be a plurality of analytes derived, may be a plurality of specimens from the same tissue isolated from a different organism, also different sites (e.g., within the same organization , or a plurality of specimens from lesions and non-lesions such as tumors).

[0045]

　In the method of the present invention, adding a certain amount of standard for a certain amount of the specimen. The unit of the amount in this case is not limited in particular, be a weight, it may be a volume. Further, when adding a solution of a standard nucleic certain amount, the unit of measuring the quantity of solution of the standard substance, the weight, volume, etc. moles may be any unit. As a method of measuring the amount of standard, Spectrophotometry, electrophoresis, column method can take various known methods such as capillary electrophoresis.

[0046]

　Before extracting nucleic acid, a given amount of a sample, adding a standard fixed amount. In the present invention, by mixing the sample to the extraction solution, after inactivating the nuclease that may be present in the specimen due guanidinium salts, it is preferable to add a standard, before separating the solution containing the RNA it is preferably added.

[0047]

　Standard materials may be added in a solid state which is dried also be added in solution with respect to the sample, but to add a certain amount exactly is preferably added in solution, several [mu] L ~ several hundred it is preferably added in an amount of [mu] L. When added in solution, is usually used pipette, it is difficult to accurately weigh and less than μL order, when it more than mL orders, capacity increases for the analyte, since the composition of the extraction solution is greatly changed, then there is a concern that an effect on the extraction operation. In the case of producing a solution of a standard substance of this case, it is preferable that the solvent is to use a buffer solution such as water or PBS.

[0048]

　The amount of standard that is added, are preferably added to the final sample to a concentration comparable to the target small RNA contained in the sample. Small RNA is concentration in the type of analyte different, if the analyte is a bodily fluid, on a molar basis, a z (zepto) mol / mL ~ p (pico) mol / mL, more preferably a ( atto) at a concentration of mol / mL ~ f (femto) mol / mL, the addition of standard for the analyte. The measurement method Spectrophotometry of its concentration, fluorescence, electrophoresis, column method, such as capillary electrophoresis method.

[0049]

　Further, Spectrophotometry after nucleic acid extraction, fluorescence, electrophoresis, column method, by measuring the extraction solution with such capillary electrophoresis, confirm the presence of the target small RNA and the standard substance, or, the amount it may be measured.

[0050]

　In the present invention, the presence of a standard substance, the extraction processing of a nucleic acid containing the target small RNA from each sample is carried out (extraction step). The standard substance was added to each sample is also extracted with the target small RNA, the nucleic acid sample obtained from each sample in the extraction step, targeted small RNA and standards.

[0051]

　How to extract nucleic acids from a sample, may be used various known methods, AGPC method generally used as a method of extracting RNA, it is preferred to use a phenol / chloroform method. In that case, preferably, the extraction solution is preferably a solution comprising 2 ~ 5M guanidine and 40-60% of the phenol. Further, as the extraction solution contaminants can be effectively removed, such as proteins, based on the total amount of the extraction solution
of (a) 50% by volume excess phenol,
(b) the total amount of solution 3 to 10 vol% of a multi
Esters, (c) a guanidinium salt of 0.5 ~ 2.0 M concentration with respect to the total amount of the
solution, (d) thiocyanate of 0.1 ~ 0.5M concentration with respect to the total amount of the solution, and
(e) buffering agents, to maintain the pH of the solution to 4-6
, it is preferred to use an extraction solution comprising a. Moreover, such a nucleic acid is extracted easily, various salts may be added to the extraction solution.

[0052]

　The method Specific extraction process, for example, by homogenizing the sample in the extraction solution to form a homogenate, which after the organic solvent for separating the aqueous solution containing the RNA was added to the homogenate, it centrifugation It can be applied. In such a process, as described above, after mixing with the solution extracted analyte, it is preferable to add the standard to before centrifugation.

[0053]

　The solution containing the extracted compact RNA, the precipitates, chromatography, centrifugation, electrophoresis, and may be purified and subjected to steps such as affinity separation. For example, in the precipitation step may be applied to step to precipitate small RNA by adding a lower alcohol to a solution containing a small RNA, collecting the precipitated small RNA. The chromatography step, the solution to a small RNA was precipitated by the addition of lower alcohols include small RNA, carrier capable of adsorbing the RNA, after adsorption to as silica membrane column and eluted from the carrier (column) recovering may be applied.

[0054]

　In the method of the present invention, to measure the amount of target small RNA and standards contained in the nucleic acid sample extracted from a plurality of specimens by the above extraction step (measurement step).

[0055]

　The measurement method is, PCR method, amplification of the sequence method, Northern hybridization method, Southern hybridization method, various methods such as hybridization of the array method and the like. Among hybridization method, it is preferable to perform the measurement by the array method using an array chip such as a microarray immobilized probes that specifically bind to target RNA and standard on a support. Specifically, it can be preferably used an array chip comprising a support and a target small RNA capture probe and the standard capture probe is aligned immobilized.

[0056]

　The "capture probe" or "probe for capturing", directly or indirectly, a nucleic acid such as RNA of the acquisition target, preferably directly, and means a substance capable of selectively binding, typically as an example, mention may be made of nucleic acids, proteins, sugars and other antigenic compounds. In the present invention, it can be preferably used a nucleic acid probe. The nucleic acid probes In addition to DNA and RNA, can be prepared by using a nucleic acid derivative such as PNA (peptide nucleic acid), or LNA (Locked Nucleic Acid). Here, the nucleic acid derivative, labeled derivatives due fluorophore, modified nucleotides (e.g., halogen, alkyl such as methyl, alkoxy such as methoxy, thio, nucleotides include groups such as carboxymethyl, and reconstruction of a base, double bond saturation, means deamination, the derivative comprising a nucleotide, etc.) that received such substitution of sulfur molecules oxygen molecules.

[0057]

　Base length of nucleic acid probes, in order to ensure the hybridization stability, it is preferably 20 bases or more. Usually, 20 if the chain length of about ~ 100 bases, the probe can be sufficiently exhibited selective binding to RNA of interest. Such chain length short oligo nucleic acid probes can be readily prepared by well known chemical synthesis method.

[0058]

　Nucleic acid probe, it is preferable to subject the nucleic acid (calibrator is the target small RNA or nucleic acid) and completely complementary nucleotide sequence, even if there is difference in a part, the target nucleic acid under stringent conditions in if high nucleotide sequence homology to the extent capable of hybridizing, it can be used as capture probes.

[0059]

　Hybridization time of stringency, temperature, salt concentration, chain length of the probe, is known to be a function of the concentration of the chaotropic agent in GC content and hybridization buffer nucleotide sequence of the probe. Stringent conditions, for example, "Sambrook, J. et al, Molecular Cloning:. (. 2nd ed) A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1998)," the like conditions described in be able to. Stringent temperature conditions is about 30 ° C. or higher. Other conditions, hybridization time, detergents (e.g., SDS) concentration, and a presence or absence of carrier DNA and the like, by combining these conditions, it is possible to set various stringency. Those skilled in the art can appropriately determine the conditions under which nucleic acid probes prepared for detection of standard that target small RNA or used contained the desired analyte can properly function as capture probes.

[0060]

　Sequence information of the small RNA can be obtained from databases such as GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Furthermore, knowledge of the miRNA sequences, for example available from miRbase website (http://www.mirbase.org/ftp.shtml). Small RNA capture nucleic acid probes can be designed based on sequence information available from those sites.

[0061]

　The number of small RNA capture probe that is immobilized on a support is not particularly limited. For example, the known sequences have been identified the number of small RNA capture probe to cover all miRNA with those immobilized on a support, may be used to determine the amount of expression of small RNA, the desired may be used as immobilized capture probes for the number of small RNA on a support, for example, it may be used a specific one or a plurality of small RNA capture probe associated with a particular disease or biological state.

[0062]

　Probe for capturing the reference material may be any probe that can be complementary capture the standard to be used. Homology with the nucleotide sequence of the reference material is 50% or more, and preferably does not take conformation, for example, can be designed in such a way are described in JP-A-2011-239708 .

[0063]

　The support capture probe is immobilized, it is possible to use the same support that is used in known microarray and microarray or the like, can be used, for example slide glass, membranes, beads and the like. Are described in Japanese Patent No. 4244788, etc., may be used a support shape having a plurality of protrusions on the surface. The material of the support is not particularly limited, glass, ceramics, inorganic materials such as silicon; can be exemplified polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate, and the like polymers such as silicone rubber.

[0064]

　As a method for immobilizing the capture probe to the support, a method of synthesizing oligo DNA on the support surface, there is known a method of dropwise oligo DNA which had been previously synthesized to a support surface fixed.

[0065]

　As the former method, Ronald et al. (U.S. Pat. No. 5705610), Michel et al. (U.S. Pat. No. 6,142,266), and Francesco et al. (U.S. Pat. No. 7,037,659) is . Since an organic solvent is used during the DNA synthesis reaction in these methods, it is desirable that the support is a material which is resistant to organic solvents. Further, in the Francesco et al. Method, for controlling the DNA synthesis from the back surface of the support is irradiated with light, the support is preferably a material having a light transmitting property.

[0066]

　As the latter method may be a method of using the methods and spotter Hirota et al (Japanese Patent No. 3922454). As a method of spot, pin system by mechanical contact of the pin tip to the solid phase, an ink jet method using the principle of an ink jet printer, capillary method and the like by capillary. After spotting process, cross linking by UV radiation, post-processing such as blocking of the surface is carried out if necessary. For immobilizing oligo DNA covalently bonded to the surface-treated substrate surface, the end of the oligo DNA is introduced functional group such as amino group or SH group. Surface modification of the support is usually carried out by treatment with a silane coupling agent having an amino group.

[0067]

　Capture probes, for one type of small RNA or calibrators may be detected by fixing a plurality of fixing areas of the support. For example, one of from the same capture probe to capture the small RNA or standard may be fixed at a plurality of locations on the support, also a type of small RNA or plural kinds of capture probes with respect to standards If you can design it may be fixed a plurality of capture probes to the same small RNA or standard targets on the support.

[0068]

　Hybridization with the probe immobilized on a support, to bind the labeled substance in the nucleic acid samples extracted from the sample prepared by adding at least one standard, the labeled nucleic acid sample was prepared with a labeling substance, the labeled nucleic acid samples are carried out by contacting the probe. In the present invention, the term "nucleic acid sample", in addition to the RNA extracted from the specimen, cDNA and cRNA prepared by reverse transcription reaction from the RNA and the like. Thus, the labeled nucleic acid sample, to may be one in which the target small RNA and standards of nucleic acid sample is labeled with a directly or indirectly labeled substance, also, cDNA prepared from RNA in the nucleic acid sample or cRNA (if the standard material is the RNA, cDNA or cRNA obtained by reverse transcription from a target small RNA and standards include) may be those labeled with directly or indirectly labeled substance.

[0069]

　As a method for binding a labeling substance to the nucleic acid sample, 'method of binding a labeling substance to the end, 5' 3 of the nucleic acid sample to the end method of binding a labeling substance, a method of incorporating the nucleotide-labeled substance is bound to the nucleic acid it can be mentioned. 3 'method of binding the terminal labeling substance, 5' in the method of binding the terminal a labeling substance may be an enzyme reaction. The enzymatic reaction may be used T4 RNA Ligase and Terminal Deoxitidil Transferase, and Poly A polymerase. Any of the labeling method also "Shao-Yao Ying ed, miRNA experimental protocol, YODOSHA, 2008," the methods that are described in can be referred to. Also, a kit for binding directly or indirectly labeled substance to the end of the RNA are various commercial. For example, a kit to bind directly or indirectly labeled substance to the 3 'end, miRCURY miRNA HyPower labeling kit (Ekishikon Co.), NCode miRNA Labeling system (Life Technologies), FlashTag Biotin RNA Labeling Kit (Genis Sphere Inc.) it can be exemplified, and the like. Life Technologies NCode miRNA Labeling system for, after addition of the poly A tail to the miRNA, and ligated to the 3 'end of the capture sequences using crosslinking oligo dT, which after hybridization to the array, the capture sequence It was added to the labeling substance having the hybridizing sequences are those that labeling miRNA through capture sequence, is a technique to bind indirectly labeled substance miRNA. 3 'end it is possible to perform labeling by these methods by using the RNA phosphorylated as a standard.

[0070]

　In addition, as in the conventional method, by synthesizing the cDNA or cRNA from RNA extracted from the specimen in the presence of the labeled deoxyribonucleotide or labeled ribonucleotide, a cDNA or cRNA labeled substance was incorporated was prepared, This is probe hybridized on the array, a method can also be called. In the case of using the RNA as a standard it is possible to adopt this method.

[0071]

　In the present invention, uses a plurality of analytes, may be used the same labeling substance either, it may be used a plurality of different labeling substances.

[0072]

　The labeling substance that can be used in the present invention, mention may be made of various labeling substances are also used in the known microarray analysis. Specifically, fluorescent dyes, phosphorescent dyes, enzymes, radioisotopes, and the like, without limitation. Preferred are measurements simple, it is detected that the signal easily fluorescent dye. Specifically, cyanine (Cyanine 2), aminomethyl coumarin, fluorescein, indocarbocyanine (Cyanine 3), cyanine 3.5, tetramethylrhodamine, rhodamine Red, Texas Red, indocarbocyanine (Cyanine 5), cyanine 5.5, cyanine 7, but include known fluorescent dyes such as oysters, but are not limited to.

[0073]

　As the labeling substance may be a semiconductor fine particles having a light-emitting properties. Such semiconductor microparticles, such as cadmium selenide (CdSe), cadmium telluride (CdTe), indium gallium phosphide (InGaP), and the like silver indium zinc sulfide (AgInZnS).

[0074]

　As described above are labeled, the nucleic acid sample containing a nucleic acid (target small RNA) and standards derived specimen is contacted with the probes on the support is hybridized. The hybridization step can be performed in exactly the same manner as conventional. The reaction temperature and time may be appropriately selected depending on the chain length of the nucleic acids being hybridized, in the case of nucleic acid hybridization is usually 1 minute to ten hours at about 30 ° C. - 70 ° C.. It performs hybridization, after washing, detecting the signal intensity from the label material in the individual probe immobilization region on a support. Detection of the signal intensity is performed using a suitable signal reader according to the type of labeling substance. In the case of using the fluorescent dye as a labeling substance may be used a fluorescence microscope and fluorescence scanner or the like.

[0075]

　Detected measurement values ​​(signal values) is compared with the ambient noise. Specifically, the measurements obtained from the probe immobilization region, comparing the measurements obtained from other positions, and detected a case where the former numerical values ​​are exceeded (valid determination positive) .

[0076]

　If the detected measurement values ​​are included background noise may be subtracted background noise. The ambient noise as the background noise can be subtracted from the detected signal values. Other, it may be used methods described in "Microarray data statistical analysis protocol (Yodo-sha)".

[0077]

　The above, the amount of target small RNA and standards present in each nucleic acid sample, i.e. the measured value of the extracted amount of the expression level and standards of the target small RNA in each sample, is obtained as a signal intensity.

[0078]

　In the method of the present invention, then, for each sample, from the extracted measurements of standard abundance of a nucleic acid sample to obtain a representative value (the representative value acquiring step). Incidentally, the term standard abundance of a nucleic acid sample, the term standard extraction amount from the analyte are synonymous in the present specification, the term "standard material abundance," "standard extraction amount" used in the same meaning. Standard abundance of a nucleic acid sample, and the standard amount extracted from the analyte, may be simply referred to as "standard amount". That is, in this specification, such as "standard amount of analyte", rather than the standard amount that is added to the sample, it means a standard extraction amount from the standard abundance or analyte in a nucleic acid sample.

[0079]

Although standards are one case of the measurement of the abundance of that one may be a representative value, in the case of two or more kinds may obtain a representative value in the following various methods.

[0080]

　Typical examples of the representative value include a mean and median. Average value means the measured value of the extracted amount of the plurality of standards (for example, measured values ​​of the signal intensities obtained using microarray) average value calculated from the. Median means measurement of the extracted amount of the plurality of standards (for example, measured values ​​of the signal intensities obtained using microarray) median obtained from.

[0081]

　These mean or median value may be a mean or median was represented by logarithm. The "to-average value expressed by a numerical value", measured value of the extracted quantity of the plurality of standards (e.g., measured values ​​of the signal intensities obtained using microarrays) logarithm of the bottom is converted to 2 logarithm in means the average value obtained. The "pair median represented by a numerical value", the measured value of the extracted amount of the plurality of standards (e.g., measured values ​​of the signal intensities obtained using microarrays) logarithm of the bottom is converted to 2 logarithm median, or a plurality of bottom central value of the measured values ​​of the extracted amount of the standard substance means a logarithm converted into 2 logarithm of. For median, it is subjected to logarithmic conversion of the measured value after be performed first the same value is obtained.

[0082]

　Mean and median, it may be one obtained by using all the measured values ​​of a plurality of standard that was measured, the measured values ​​of some elected from among the standard of the plurality of or it may be obtained by using. For example, may be obtained by using all of the measurements taken in standard capture probe mounted on a microarray, a part of the standard capture probe (e.g., acquisition standards mounted on a microarray When the probe assumed to be 10 or may be determined for five) of them. For example, it is possible to elect only standard capture probe was in effect determined positive common to all specimens should be comparative analysis to obtain a representative value of the standard substance. Further, prior to finding a representative value, it may exclude outliers from the measured abundance of standards.

[0083]

　Further, by detecting using one of the array or using a plurality of capture probes with respect to standards, or one type of capture probe was spotted at a plurality of locations, a plurality for a single standard in some cases where the measured value can be obtained. In this case, as in the case of using two or more kinds of standards, be used an average value calculated from a plurality of measured values ​​or median, the mean or median was represented by example logarithm as a representative value it can. Further, it may also be determined representative value using all those measurements may be determined using a part of the measurement values.

[0084]

　Further, in embodiments using a plurality of standards, one type of capture probe to one of the standard substance if it is spot at a plurality of locations on the array, respectively, is first signal measurement from the plurality of capture probe spots per standard the average value of the values, each average log transformed, may be an average value or median between multiple standards with each logarithm. Mean or median was determined in this manner are also encompassed by the "mean or median was represented by logarithm."

[0085]

　CV value of the representative values ​​in the plurality of samples (coefficient of variation) is preferably of 0.5 or less. Usually, CV value of the measured values ​​in the case of using the microarray is 0.5 or less. CV value is large variation in the case of more than 0.5, the extraction efficiency of the standard substance in the extraction step is not stabilized, the accuracy of the data after the correction as a result is expected to decrease.

[0086]

　Subsequently, the representative value of the standard extraction of each sample obtained by the representative value acquiring step, using the arbitrarily set reference value with respect to standard extraction amount, expression of a target small RNA obtaining a correction coefficient used for correction of the amount (correction coefficient acquisition step). In the correction coefficient acquiring step, it may be a correction coefficient is obtained using the difference between the representative value and the reference value may be obtained correction coefficient by using a ratio between the representative value and the reference value. Is not particularly limited, it is used to determine the correction coefficient by using a difference in the case when using a representative value which is not logarithmic conversion obtains the correction coefficient by using the ratio, using a representative value expressed in log preferable. Hereinafter, processes for these two types.

[0087]

　correction coefficient acquisition step -1 is a method of utilizing a difference between the representative value and the reference value of the standard extraction amount. In this step, the following 1-1. Reference analyte acquisition method, or 1-2. It may apply a fixed value correcting method.

[0088]

1-1. Reference analyte acquisition method
　arbitrarily selects one sample (first sample) from a plurality of analytes to be analyzed, it is the "reference sample". The remaining one or more analytes (analyte second later), the "analyte to be corrected."

[0089]

　In this specification, the term "second and subsequent analyte" also encompasses the second analyte. For example, if the two are more analytes to be compared, the analyte to be corrected is only a second analyte, if a plurality of analytes in three should be compared, the analyte to be corrected second sample and the two are in the third sample.

[0090]

　In this way, the representative value of the standard substance of the reference sample is a "reference value". And the reference value, the difference between the representative value of the standard amount of each analyte in the second and subsequent (analyte to be corrected), is used as a correction factor for the respective sample of the second and subsequent. Correction factor will be acquired by the number of analytes to be corrected.

[0091]

　Specifically, the correction coefficient is calculated by Equation 1 or 1 '.
　c 1-1 = (representative value of the standard substance of the reference sample (standard value))
　　　　- (corrected by the representative value of the standard amount of analyte) Equation 1
　c 1-1 analyte to be '= (correction representative value of the amount of standard)
　　　　　- (standard amount of the representative value of the reference sample (standard value)) equation 1 '

[0092]

　For example, measurements were performed of the expression amount using the microarray, the case of using an average value expressed by logarithm as a representative value of the standard amount, the correction coefficient for the analyte to be corrected, Formula 2 or Formula 2 ' it can be obtained at.

[0093]

[Number 1]

[0094]

[Number 2]

[0095]

　Here, in Equation 2, Equation 2 ',
　n is the total number of standard capture probe immobilization region on the support,
　Aj is the reference analyte, standard capture probe fixed j-th (1 ≦ j ≦ n) signal measurements from regions,
　Xj is the second analyte, j-th signal measurements from standard capture probe immobilization regions (1 ≦ j ≦ n),
is.

[0096]

　When the probe is standard one-to-one correspondence, n is standard capture probe on the support is equal to the number of standard that becomes a target.

[0097]

　'In, instead of n, the total number n of the standard substance capturing probe immobilization region that was in effect determined positive common to all the samples to be compared' Equations 2 and 2 can be used.

[0098]

1-2. Fixed value correcting method
　for a representative value of the standard amount, it is previously assumed way as to take a certain numerical in all specimens. That is, a fixed numerical value and a "reference value", with the difference between the representative value of the standard amount of the fixed number and each specimen, utilizing this difference as a correction coefficient. In this method, 1-1. Since the "reference sample" is not present as shown in, next all of the plurality of analytes to be compared analyzed is "specimen is corrected" so that the correction coefficient by the number of specimens to be compared analyzed is obtained.

[0099]

　Specifically, the correction coefficient is calculated by Equation 3 or Equation 3 '.
　r 1-2 = (fixed numeric value (reference value)) - (corrected by the representative value of the standard amount of analyte)
　　　　　　　　　　　　　　　　　　　　　　　　　　　　Equation 3
　r 1-2 '= (corrected by standard amount of value representative of the analyte ) - (fixed number (reference
　　　　　　　　　　　　　　　　　　　　　　　　　　　　value)) equation 3 '

[0100]

　For example, measurements were performed of the expression amount using the microarray, the case of using an average value expressed by logarithm as a representative value of the standard amount, the correction coefficient for the analyte to be corrected, Formula 4 or Formula 4 ' it can be obtained at.

[0101]

[Number 3]

[0102]

[Formula 4]

[0103]

　Here, Equation 4, wherein 4 ',
　alpha is a reference value (fixed numeric), n
　is the total number of standard capture probe immobilization region on the support,
　Yj is the analyte, j-th (1 ≦ j ≦ signal measurements from standard capture probe immobilization regions n),
it is.

[0104]

　When the probe is standard one-to-one correspondence, n is standard capture probe on the support is equal to the number of standard that becomes a target.

[0105]

　'In, instead of n, the total number n of the standard substance capturing probe immobilization region that was in effect determined positive common to all the samples to be compared' Equations 2 and 2 can be used.

[0106]

　Fixed numbers used as a reference value at a fixed value correcting method, in at least one of comparative analysis, as long as using the same numerical consistently for all analytes may use any number (but non-zero) . Using the same expression measurement system, and is always if it uses the same numbers as the fixed numbers, can be compared also analyzed among specimens were measured for the expression level for another day. For example, the amount of each standard substance added to the sample are identical in all the samples, it may determine the fixed numerical value based on the standard amount. However, the magnitude of the signal value detected by the system used in the measurement process for may vary, the fixed numerical depending on the system to be used can be freely selected.

[0107]

　correction coefficient acquiring step-2 is a method of utilizing the ratio between the representative value and the reference value of the standard amount. In this step, following 2-1. Reference analyte acquisition method, or 2-2. It may apply a fixed value correcting method.

[0108]

2-1. Reference analyte acquisition method
　arbitrarily selects one sample (first sample) from a plurality of analytes to be analyzed, it is the "reference sample". The remaining second and subsequent sample, the "analyte to be corrected."

[0109]

　In this way, the representative value of the standard substance of the reference sample and a "reference value", and the reference value, the ratio between the representative value of standard amount of each analyte in the second and subsequent (analyte to be corrected), used as a correction factor for the respective sample of the second and subsequent. Correction factor will be acquired by the number of analytes to be corrected.

[0110]

　Specifically, the correction coefficient is calculated by Equation 5 or Equation 5 '.
　c 2-1 = (representative value of the standard substance of the reference sample (reference value))
　　　　　/ (corrected by the representative value of the standard amount of analyte) Equation 5
　c 2-1 analyte to be '= (correction representative value of the standard amount)
　　　　　　/ (standard amount of the representative value of the reference sample (standard value)) equation 5 '

[0111]

　For example, measurements were performed of the expression amount using the microarray, the case of using an average value expressed by logarithm as a representative value of the standard amount, the correction coefficient for the second analyte, wherein 6 or Formula 6 ' it can be obtained at.

[0112]

[Formula 5]

[0113]

[Number 6]

[0114]

　Here, Formula 6, wherein 6 ',
　n is the total number of standard capture probe immobilization region on the support,
　Aj is the reference analyte, standard capture probe fixed j-th (1 ≦ j ≦ n) signal measurements from regions,
　Xj is the second analyte, j-th signal measurements from standard capture probe immobilization regions (1 ≦ j ≦ n),
is.

[0115]

　When the probe is standard one-to-one correspondence, n is standard capture probe on the support is equal to the number of standard that becomes a target.

[0116]

　'In, instead of n, the total number n of the standard substance capturing probe immobilization region that was in effect determined positive common to all the samples to be compared' Formula 6 and Formula 6 may also be used.

[0117]

2-2. Fixed value correcting method
　for a representative value of the standard amount, it is previously assumed way as to take a certain numerical in all specimens. That is, a fixed numerical value and a "reference value", with the ratio of the representative value of the standard amount of the fixed number and each specimen, utilizing this ratio as a correction coefficient. In this method, 2-1. Since the "reference sample" is not present as shown in, next all of the plurality of analytes to be compared analyzed is "specimen is corrected" so that the correction coefficient by the number of specimens to be compared analyzed is obtained.

[0118]

　Specifically, the correction factor is determined by Equation 7 or Equation 7 '.
　r 2-2 = (fixed numeric value (reference value)) / (corrected by the representative value of the standard amount of analyte)
　　　　　　　　　　　　　　　　　　　　　　　　　　　　Equation 7
　r 2-2 '= (corrected by standard amount of value representative of the analyte ) / (fixed numerical value (reference
　　　　　　　　　　　　　　　　　　　　　　　　　　　　value)) equation 7 '

[0119]

　For example, measurements were performed of the expression amount using the microarray, the case of using an average value expressed by logarithm as a representative value of the standard amount, the correction coefficient for the analyte to be corrected, wherein 8 or Formula 8 ' it can be obtained at.

[0120]

[Number 7]

[0121]

[Number 8]

[0122]

　Here, Formula 8, wherein 8 ',
　alpha fixed numeric,
　n is the total number of standard capture probe immobilization region on the support,
　Yj is standard in the specimen, j-th (1 ≦ j ≦ n) signal measurements from material capture probe immobilization region,
a.

[0123]

　When the probe is standard one-to-one correspondence, n is standard capture probe on the support is equal to the number of standard that becomes a target.

[0124]

　'In, instead of n, the total number n of the standard substance capturing probe immobilization region that was in effect determined positive common to all the samples to be compared' Equation 8 and Equation 8 can also be used.

[0125]

　As used herein, a reference value of the "fixed numerical" Details are similar to fixed numbers in "1-2. Fixed-value correcting method".

[0126]

　Next, using the correction coefficient obtained by the correction coefficient acquiring step 1 or the correction coefficient acquiring step-2, using the method of correction steps -1 or correction step-2, in a specimen to be corrected correcting the expression level of the target small RNA.

[0127]

　correction process -1 is a method of correcting the expression level of the target small RNA using the correction coefficient obtained by the correction coefficient acquiring step -1, the correction factor to the expression level of the target small RNA addition, or corrects by subtracting the correction factor from the expression level. In this step, there is a correct two ways, each of which corresponds to the reference sample acquisition method of correction coefficient acquisition step -1 to a fixed value correcting method.

[0128]

1-1. Reference analyte acquisition method
　correction in the expression level of the target small RNA in the second and subsequent sample is performed using a correction coefficient for the second and subsequent sample, respectively. That is, when correcting the expression level of a target small RNA for the second analyte correction coefficient (c2 for the second analyte 1-1 or c2 1-1 using '), a third sample in correcting the target miRNA expression level correction coefficient (c3 for the third analyte 1-1 or c3 1-1 using a ').

[0129]

　As a correction factor, when using a difference obtained by subtracting the representative value of the standard amount of the second and subsequent sample from the representative value of the standard amount of the reference sample, i.e. in the case of the formula 1, each of the specimens in the second and subsequent by adding the correction factor to the logarithm of the measured values ​​or measured value of the target small RNA expression levels, the second and subsequent correction of the expression level of a target small RNA for each sample is performed. Expressing correction in this case the formula is corrected expression level Ei of the i-th target small RNA in the "analyte to be corrected" is calculated according to the following formula 9.

[0130]

[Number 9]

[0131]

　Here, Wi is signal measurement value from the i-th small RNA capture probe immobilization region.

[0132]

　On the contrary, as the correction coefficient, from the representative value of the standard substance of the second and subsequent sample, when using a difference obtained by subtracting the representative value of the standard amount of the reference sample, i.e. in the case of the formula 1 ' , by subtracting the correction factor from the logarithm of the measured values ​​or measured value of each target small RNA expression level in the specimen of the second and subsequent, second and subsequent correction of the expression level of a target small RNA for each sample is carried out It is. Expressing correction in this case by the formula, the corrected expression level Ei of the i-th target small RNA at a "sample to be corrected" can be determined by the following equation 9 '.

[0133]

[Formula 10]

[0134]

　Here, the definition of Wi are the same as above formula 9.

[0135]

　If the corrected target small RNA expression levels measured in the second sample, or added to c2 to the logarithm of the measured values ​​or measured value of each target small RNA expression in the second sample, or the c2 'should be subtracted. The same applies to the sample of the third and subsequent. The calculation that is the difference between the representative value and the reference value of the first sample on the basis analyte is naturally 0, the structure of the program, adding or subtracting the 0 target small RNA expression level of the first sample no problem be carried out.

[0136]

1-2. Fixed value correcting method
　correction target small RNA expression levels is performed using the correction coefficient obtained by the difference between the representative value and a fixed numeric value (reference value), respectively. That is, when correcting the target small RNA expression levels for a specimen, the correction coefficient (r for the sample 1-2 or r 1-2 using a ').

[0137]

　As a correction factor, when using a difference obtained by subtracting the representative value of the standard amount of the specimen from a fixed number, that is, when the above equation 3, pairs of measured values ​​or measured value of the expression level of each target small RNA in a specimen by adding the correction factor to a number, the correction of the expression level of a target small RNA for each sample is performed. Expressing correction in this case the formula is corrected expression level Ei of the i-th target small RNA in the "analyte to be corrected" can be determined by the following equation 10.

[0138]

[Number 11]

[0139]

　Here, Wi is signal measurement value from the i-th small RNA capture probe immobilization region.

[0140]

　On the contrary, as the correction coefficient, when using a difference obtained by subtracting the fixed number from the representative value of the standard amount of the analyte, that is, when the above formula 3 'is a measure of each target small RNA expression in the sample or by subtracting the correction factor from the measured value of the logarithm, the correction of the target small RNA expression levels for each sample is performed. Expressing correction in this case by the formula, the corrected expression level Ei of the i-th target small RNA at a "sample to be corrected" is calculated according to the following formula 10 '.

[0141]

[Number 12]

[0142]

　Definition of Wi is the same as in the above formula 10.

[0143]

　correction step-2 is a method of using the correction coefficient obtained by the correction coefficient acquiring step-2 to correct the expression level of the target small RNA, the correction factor the expression level of a target small RNA in division, or it corrects by multiplying the correction coefficient to the expression level. Also in this step, there is a correct two ways, each of which corresponds to the reference sample acquisition method of correction coefficient acquiring step-2 to the fixed-value correcting method.

[0144]

2-1. Reference analyte acquisition method
　correction in the expression level of the target small RNA in the second and subsequent sample is performed using a correction coefficient for the second and subsequent sample, respectively. That is, when correcting the expression level of a target small RNA for the second analyte correction coefficient (c2 for the second analyte 2-1 or c2 2-1 using '), a third sample in correcting the target small RNA expression level correction coefficient (c3 for the third analyte 2-1 or c3 2-1 using a ').

[0145]

　As the correction coefficient, when the representative value of the standard substance of the second and subsequent sample as the denominator to be corrected, using a ratio of the representative value of the standard amount was molecule reference analyte, that is, when the above equation 5, by multiplying the correction coefficient to the logarithm of the measured values ​​or measured value of each target small RNA expression level in the specimen of the second and subsequent, second and subsequent correction of the expression level of a target small RNA for each sample are carried out that. Expressing correction in this case the formula is corrected expression level Ei of the i-th target small RNA in the "analyte to be corrected" is calculated according to the following formula 11.

[0146]

[Formula 13]

[0147]

　Here, Wi is signal measurement value from the i-th small RNA capture probe immobilization region.

[0148]

　On the contrary, as the correction coefficient, a representative value of a standard substance of the reference sample as the denominator, in the case of using the ratio in which the representative value of the standard amount of the second and subsequent analytes and molecules, i.e. the formula 5 ' in this case, by dividing the correction coefficient logarithm of the measured values ​​or measured value of each target small RNA expression level in the specimen of the second and subsequent, the second and subsequent expression of the target small RNA for each sample correction is carried out. Expressing correction in this case by the formula, the corrected expression level Ei of the i-th target small RNA at a "sample to be corrected" is calculated according to the following formula 11 '.

[0149]

[Number 14]

[0150]

　Here, the definition of Wi are the same as the formula 11.

[0151]

　If the corrected target small RNA expression levels measured in the second sample, c2 to the logarithm of the measured values or measured value of each target small RNA expression in the second sample 2-1 dividing or c2 2-1 may be multiplied by a '. The same applies to the sample of the third and subsequent. In the procedure of Equation 5 and Equation 11, in the procedure of Equation 5 'and Formula 11', the value of the expression level Ei of the finally obtained corrected target small RNA is identical. Although the ratio of the fixed numerical (reference value) in the representative value with the method of the first analyte and the reference analyte is naturally 1, the structure of the program, the target small RNA expression level of the first sample 1 no problem be carried out calculations that divide by multiplication or target small RNA expression levels 1.

[0152]

2-2. Fixed value correcting method
　correction target small RNA expression levels is performed using the correction coefficient obtained by the ratio of the fixed numerical (reference value), respectively. That is, when correcting the target small RNA expression levels for a specimen, the correction coefficient (r for the sample 2-2 or r 2-2 using a ').

[0153]

　As a correction coefficient, a representative value of standard amount of analyte as the denominator, the case of using the ratio of the fixed numerical molecular, that is, in the case of Equation 7, the measurement value of the expression level of each target small RNA in a specimen or the by multiplying the correction coefficient to the logarithm of the measured values, the correction of the expression level of a target small RNA for each sample is performed. Expressing correction in this case the formula is corrected expression level Ei of the i-th target small RNA in the "analyte to be corrected" can be determined by the following equation 12.

[0154]

[Number 15]

[0155]

　Here, Wi is signal measurement value from the i-th small RNA capture probe immobilization region.

[0156]

　On the contrary, as the correction coefficient, a representative value of standard amount of analyte as the denominator, the case of using the ratio of the fixed numerical molecular, that is, when the equation 7 ', each target small RNA expression in the sample by dividing the logarithm of the measured values ​​or measured value of the amount by the correction factor, the correction of the target small RNA expression levels for each sample is performed. Expressing correction in this case by the formula, the corrected expression level Ei of the i-th target small RNA at a "sample to be corrected" is calculated according to the following formula 12 '.

[0157]

[Number 16]

[0158]

　Here, the definition of Wi are the same as above formula 12.

[0159]

　Using the corrected target small RNA expression levels, contrasting target small RNA expression levels between a plurality of analytes. When the correction by the reference sample acquisition method is carried out, since the target small RNA expression level of the first analyte and the reference analyte has not undergone correction, between for example the first sample and the second sample contrast is the target small RNA expression level of the first sample that has not been corrected, but the contrast between the target small RNA expression levels of the corrected second sample, at least one of the analytes to be compared One will be always corrected specimen. Therefore, "the corrected target small RNA expression levels, contrasting target small RNA expression levels between a plurality of bodily fluid analyte" if such is between uncorrected reference analyte and corrected other analytes manner of contrasting are also included.

[0160]

　Comparative analysis process itself can be performed as in the conventional method. For example, it is sufficient to create a scatter plot of the expression level data called scatter plot. If the sample to be compared is three, for example, two of the scatter plot comparing analyzed and any one specimen and each remaining sample out of three (e.g., first analyte - between the second sample scatter plots and first analyte - may be scatter plot) created between the third analyte, if necessary, when further among the remaining two specimens (in the preceding example, further the 2 specimens - may be created scatter plot comparative analysis at the inter-3 specimens). Comparative analysis of the four or more analytes may also be carried out in the same manner. Incidentally, if the comparative analysis of 3 samples, it is also possible to create a three-dimensional scatter plot. Even if the reference sample acquisition method is not necessarily reference analyte and the other the second sample mean must be compared, respectively, for example, the second sample - comparison between the reference sample and the second sample - the third comparison between samples may be performed.

[0161]

　Further, the corrected target small RNA expression levels, one of the sample and the difference in the expression level of the target small RNA with the rest of the other analytes calculated, logarithmized fold change with the difference (fold -change) may represent the comparative analysis result. For example, the expression level of a target small RNA in reference analyte (for reference analyte acquisition method) or the expression level of the corrected target small RNA in the first sample and (in the case of fixed-value correcting method), in the specimen of the second and subsequent the difference between the expression level of the corrected target small RNA may be calculated. As in the case of this case above, calculating the difference between the first sample and is not limited to calculating the difference between the other samples, any one other analytes of the second and subsequent sample it may be. Furthermore also uses the corrected target small RNA expression levels, average and standard deviation, standard error, the calculation of the variation coefficient, inter-group comparison and significance test, such as cluster analysis, target in a plurality of specimens it is also possible to carry out the comparison and evaluation by statistical analysis using the expression amount of small RNA.

[0162]

　The apparatus of the present invention is a device for correcting the expression level for comparative analysis of the expression level of the target small RNA. The apparatus,
　a plurality of the sample, after the addition of the at least one standard nucleic acid length is a nucleic acid of at least 200 bases was determined using a nucleic acid sample obtained by extracting the nucleic acid from the sample , storage means and for storing the measured values of the target small RNA expression levels and standards extraction amount for each sample;
　representative for each specimen, from the measured values of standard extraction amount, the representative value, which is preferably represented by logarithm to obtain the value, the representative value acquiring means;
　and optionally set reference value with respect to the amount of extracted standard, the Samata the ratio of the representative value of each sample obtained by the representative value acquiring unit, each sample respectively acquires a correction coefficient of the target small RNA expression levels for the correction coefficient acquiring means and;
　using each correction coefficient acquired by the correction coefficient acquiring section, the expression level of a target small RNA measured in the respective sample perform the correction, And a positive means
including.

[0163]

　In a certain aspect, the reference value is a representative value of the standard amount of the first specimen is arbitrarily selected (reference samples), the expression level of a target small RNA measured in the sample of the second and subsequent correction It is. That is, in this embodiment, the device of the present invention,
　the plurality of each specimen, after the nucleic acid length plus at least one reference material is 200 or more bases of a nucleic acid, obtained by extracting nucleic acid from the sample was measured using a nucleic acid sample, the storage means and for storing the measured values of the target small RNA expression levels and standards extraction amount for each sample;
　for each sample, from the measured values of standard extraction amount, the representative value, preferably obtaining a representative value expressed in logarithm value, the representative value acquiring means;
　and optionally first analyte reference sample that has been selected, a representative value of standard extraction amount of the reference sample as a reference value, the reference the Samata the ratio of the representative value of the values and the remaining second and subsequent sample, respectively acquires a correction coefficient for the second and subsequent sample, the correction coefficient acquiring means and;
　obtained by the correction coefficient acquiring section complement for the second and subsequent specimen Using coefficients For each of the measured target small RNA expression amount of the correction in the sample of the second and subsequent, and correcting means
including.

[0164]

　In another embodiment, the reference value is a fixed value that is arbitrarily defined in terms standard extraction amount, for all the samples including the first sample, is carried out to correct the target small RNA expression levels. That is, in this embodiment, the device of the present invention,
　the plurality of each specimen, after the nucleic acid length plus at least one reference material is 200 or more bases of a nucleic acid, obtained by extracting nucleic acid from the sample was measured using a nucleic acid sample, the storage means and for storing the measured values of the target small RNA expression levels and standards extraction amount for each sample;
　for each sample, from the measured values of standard extraction amount, the representative value, preferably obtaining a representative value expressed in logarithm value, the representative value acquiring means;
　as a reference value fixed numerical difference between the representative value of the reference values and the analyte ratio as the correction factor for the sample respectively acquired, a correction coefficient acquiring section;
　using each correction coefficient for each sample obtained by the correction coefficient acquisition unit corrects the expression level of the measured target small RNA in each sample, correcting means theft
, including the.

[0165]

　The comparative analysis system for a small RNA expression levels including a correction device described above, further, the corrected target small RNA expression levels, an output means for outputting a result of comparing the target small RNA expression levels between the at least two analytes It may include.

[0166]

　A block diagram schematically showing the configuration of an analysis device including the correction device shown in FIG. Analyzer 10 includes an input unit 110, display unit 120, an output unit 130, storage unit 140, the control unit 150, conversion unit 160 comprises an analysis unit 170. Further, FIG. 3 shows an example of a flowchart of a correction process of the target small RNA expression levels according to the present invention.

[0167]

　The input unit 110 is means for inputting information relating to the operation of the analysis device 10. It can be preferably used a known input means such as a keyboard. When using a microarray, data of small RNA expression levels and standard extraction amount obtained by hybridization assays, for example, the apparatus of the present invention is read by the reading means, such as another scanner, it was converted into numerical data after, the numerical data from the input unit 110 may be input to the analyzer 10. Alternatively, the reading means such as a scanner is, (not shown) which may be included in the analysis apparatus 10 of the present invention.

[0168]

　Data input expression amount and the extracted amount of data input from 110, or the expression level was quantified read by integrated reading means to the analyzer 10 and the extracted amount is stored in the storage unit 140. At this time, the storage unit 140, for each of a plurality of analytes, acts as a storage means for storing the measured value of the expression level of a plurality of target small RNA and extraction amount of at least one standard at a time.

[0169]

　Measurement data of small RNA expression level of each sample stored in the storage unit 140 and the standard extraction amount, in some cases by the conversion unit 160, the bottom is converted to the logarithm of such 2. Then, the analysis unit 170, for each sample, representative values ​​of the measured values ​​of standard extraction amount is acquired. Representative value, as mentioned in the description of the correction method, for example, at least one of an average value or median value of the standard substance amount measurement (one only standard even when used for correction, to measure it when the probe immobilization regions are a plurality present on the array, the representative value may be the average or can be a central value), or measurement of the particular one of the standard amount.

[0170]

　After the representative values ​​are acquired, the analyzing unit 170, Samata between the reference value and the representative value of the standard amount of each sample for each sample the ratio is calculated, the correction factor is obtained, respectively. Details of obtaining the correction factor is as described in correction method. In the case where the reference analyte acquisition method is adopted, the structure of the program but also (if calculating the difference) correction coefficient 0 for the first analyte which is a reference analyte or correction factor 1 (the case of calculating the ratio ) no problem be configured so as to acquire.

[0171]

　In the apparatus 10, to elect or reference analyte to enter the reference value may be performed by a person operating the apparatus 10 to specify any one sample from the input unit 110. Alternatively, it may be selected automatically reference value device 10 may elect one specimen serving as a reference sample. For example, data is input from the input unit 110, the specimen initially data in the storage unit 140 is stored can be selected as the reference sample by the apparatus 10. Step elected or input of the reference sample has been positioned after the convenience representative value acquiring step in FIG. 3 (S-3), is not limited to this, at an earlier step, for example, when storing the data it may be executed. Alternatively, it may be registered in the conversion unit 160 or the like fixed number specified in advance as a reference value.

[0172]

　Then, the analysis unit 170, a correction factor for each sample using respectively, to correct the measured target small RNA expression level data. Correction operation details are as described in correction method. In the case where the reference analyte acquisition method is adopted, the configuration of the program, for the target small RNA expression level data of the first analyte which is a reference analyte, (if calculates a difference) correction coefficient 0 or correction no problem even possible to perform the correction operation using the coefficient 1 (the case of calculating the ratio).

[0173]

　Then, a analysis unit 170, comparison and statistical analysis of a target small RNA expression level of each sample is performed. Result of comparison and statistical analysis, the output unit 130, is output to the display unit 120, is displayed. Furthermore, comparison results and statistical analysis result to the output device or a recording medium such as a printer or the like can be output. Furthermore, the output unit 130 may be configured to output a comparison analysis results and statistical analysis result to an external storage device such as a database, that exists outside of the apparatus via a network.

[0174]

　Storage unit 140, in addition to storing the measured values ​​of the plurality of expression levels of small RNA and extraction of a plurality of standards, intermediate analysis results stored appropriately produced in each step of the above.

[0175]

　Various operations described above of the device 10 is controlled by the control unit 150. Specifically, as indicated by a dotted arrow in FIG. 2, the input unit 110, display unit 120, an output unit 130, storage unit 140, the control unit 150, conversion unit 160, for each unit of the analyzing section 170, control information from the control unit 150 is output, the means based on the control information to work together, the entire device 10 operates.

[0176]

　Further, the present invention provides a program for causing a computer to function as a correction device or analyzer described above. The program, specifically, the means (i.e., the storage unit, the representative value acquiring unit, the correction coefficient acquiring means, correcting means, and, further output means in the analysis apparatus) as described above the computer program to function as it is. Alternatively, the present invention provides a program for executing the steps of the correction method or comparative analysis methods including the correction method of the present invention described above to a computer. The correction method, the measurement step described above, the representative value acquiring step, the correction coefficient acquiring step, and the correction step includes, in the comparative analysis method, further, the corrected target small RNA expression levels, among a plurality of specimens It may include comparative analysis step of comparing the target small RNA expression levels. These programs, using the data of the measured values ​​of standard substances extracted amount measured at the same time as the expression level of the target small RNA by microarray or the like, a program for executing the correction of the expression level of the target small RNA to the computer.

[0177]

　Furthermore, the present invention is recorded with the above-mentioned one of the programs, and a computer-readable recording medium.

[0178]

　"Recording medium", a flexible disk, a magneto-optical disk, ROM, EPROM, EEPROM, CD-ROM, MO, be any "fixed physical medium" such as a DVD (non-transitory recording medium). Alternatively, LAN, WAN, typified by the Internet, such as a communication line or a carrier wave when over the network to transmit the program, may be a short-term holding the program "communications media."

[0179]

　The "program" is a data processing method written in any computer language and written method, may be of any format such as source code or binary code. Note that "program" is not necessarily limited to those composed singularly, what is distributed configuration as a plurality of modules or libraries, or in cooperation with a different program such as the OS (Operating System) that also including those to achieve the function. It should be noted that the specific configuration for reading the recording medium in each device shown in the embodiment, the reading procedure or, for such installation procedure after reading, may be well-known configuration and procedure.

[0180]

　May be preferably used in the present invention, a plurality of target small RNA capture probe, at least one, preferably an array chip comprising a support and a plurality of standard capture probe immobilized, as a chip for small RNA Expression Analysis it is possible to provide. Preferred conditions for the chip is as described in the measurement process of the present invention.
Example

[0181]

　Hereinafter, the present invention will be specifically described based on the embodiment applying the correction method of the present invention with human serum specimens. However, the present invention is not limited to the following examples.

[0182]

(Standard)
　as the standard substance for example a nucleic acid length of 200 bases or more nucleic acids may be purchased from National Institute of Advanced Industrial Science and Technology as a ribonucleic acid (RNA) solution for quantitative analysis, five RNA solution: sample name RNA500-a (SEQ ID NO: 1), RNA500-B (SEQ ID NO: 2), RNA500-C (SEQ ID NO: 3) (all nucleic acid length of about 500 bases of RNA), RNA1000-a (SEQ ID NO: 4) and RNA1000-B (SEQ ID NO: 5) (all RNA nucleic acid length of about 1000 bases) entrusted synthesized in CRMs NMIJ CRM 6204-a made of, as well as the present inventors have designed euro fins Genomics Inc. 3 types of RNA, Hsncs_071028 (SEQ ID NO: 15), Hsncs_404161 (SEQ ID NO: 16), Hsncs_647981 (SEQ ID NO: 17) (all RNA nucleic acid length of about 200 bases) RNA was used for.

[0183]

　As standards for comparison example nucleic acid length is a nucleic acid of less than 200 bases, et al Nucleic Acids length indicated by 1 is 20 bases cel-miR-39 is a miRNA from a non-human (SEQ ID NO: 6) , cel-miR-54 (SEQ ID NO: 7), ath-mir-159a (SEQ ID NO: 8), was selected cel-miR-238 (SEQ ID NO: 9). For each miRNA, it was synthesized to the 5'-end Euro fins Genomics Inc. as RNA which put a phosphate group modification. Also, Euro fins are non-biological origin sold by Genomics Company nucleic length 5 kinds of RNA 60 bases, H2NC000001 (SEQ ID NO: 10), H2NC000002 (SEQ ID NO: 11), H2NC000003 (SEQ ID NO: 12), H2NC000005 (SEQ ID NO: 13), was used H2NC000006 (SEQ ID NO: 14).

[0184]

　The physical properties of each standard are shown in Table 1. For all standard materials used for all of the human gene transcripts that have been recorded in public databases BLAST, sequence homology was confirmed to be 50% or less.

[0185]

[Table 1]

[0186]

(Capture probe design)
　as the target small RNA, select 2555 one human miRNA obtained from miRBase Release 19, using a DNA having the complementary sequence as the target small RNA capture probe (http: //www.sanger. ac.uk/Software/Rfam/mirna/index.shtml).

[0187]

　The standard nucleic acid substance capturing probe for example, the RNA500-A, RNA500-B, RNA500-C, RNA1000-A, RNA1000-B, hsncs_071028, hsncs_404161, using a DNA having a complementary sequence of the sequence of Hsncs_647981.

[0188]

　As the standard capture probe for comparative examples, cel-miR-39 (22 bases) obtained from miRBase, cel-miR-54 (24 bases), ath-mir-159a (21 bases), cel-miR using a DNA having a complementary sequence of -238 (23 bases). Furthermore, using a DNA having a complementary sequence of a commercially available the five RNA (H2NC000001, H2NC000002, H2NC000003, H2NC000005, H2NC000006).

[0189]

　Capture probes of the target small RNA capture probe and standard nucleic acid material, using a 3 'synthetic DNA obtained by introducing an amino group at the end (commissioned synthesized by Euro fins Genomics Inc.).
(Preparation of DNA microarrays)
　3 'end to the amino group introduced above 2555 one target small RNA (miRNA) capture probe and total 17 kinds of standard capture probe, manufactured by Toray Industries, Inc. of "3D-Gene" (registered immobilized on the convex portion of the TM) substrate (3000 poster board) to produce a DNA microarray. The standard substance capturing probes were secured with each eight points. Using this DNA microarray was carried out the following experiment.

[0190]

(Added and extraction step of the nucleic acids of the standard substance to serum samples)
　to serum specimens 300 [mu] L, the 3D-Gene RNA extraction reagent from liquid sample ( Toray) is RNA extraction reagent mixing 900 [mu] L, in the embodiment, the above-described RNA500-a, RNA500-B is a standard for example, RNA500-C, RNA1000-a , RNA1000-B, hsncs_071028, at least one was added each 10 fmol, comparative examples of hsncs_404161 and Hsncs_647981, is a standard for comparison example described above cel-miR-39, cel- miR-54, ath-mir-159a, at least one of cel-miR-238, H2NC000001, H2NC000002, H2NC000003, H2NC000005 and H2NC000006 each was 10fmol added, by centrifugation, the upper aqueous layer was collected, RNA was purified using miRNeasy mini kit (Qiagen).

[0191]

(Expression level and measuring step of extraction of standard target small RNA by DNA microarray (miRNA))
　The obtained target small RNA (miRNA), were labeled with 3D-Gene miRNA labeling kit (Toray). Labeled miRNA, in accordance with standard protocols of 3D-Gene miRNA chip (Toray Industries, Inc.), was subjected to hybridization and washing. The reacted DNA microarray were detected fluorescent signal using a microarray scanner (Toray). Scanner settings, the laser output of 100%, the voltage setting of the photomultiplier was 42%.

[0192]

　according to the procedure described above, SEQ ID NO: 1 to Example for reference material (all five) represented by 5 or SEQ ID NO: 15 through Example for standard (all three) represented by 17 commercial was added to the serum specimens 300 [mu] L, step extraction of nucleic acids, and measurement was carried out step abundance of expression levels and standards of the target small RNA (miRNA) by DNA microarray. Above steps, in order to compare the effect of correcting the variation of expression level measurements between the measurement experiment (variation) was repeated 10 times.

[0193]

　As a result, about 1000 types of miRNA of 2555 one miRNA is detected. The median of the whole of the detected approximately 1000 types of miRNA CV value (coefficient of variation) among 10 measurements experiments expression of each miRNA of the detected approximately 1000 types, before correction 0.45 met It was.

[0194]

　Correction of the expression level of each miRNA was carried out as follows. First, the abundance of each standard in each round of experiments, calculated as an average value of the measured values ​​of the eight points on the DNA microarray was calculated as the representative value. Next, a reference value representative value of the standard substance in the first experiment, the ratio of the representative value of two to ten times of experiments were obtained as the correction coefficient. The correction of the measured value of expression amount of miRNA in the correction factor two to ten times of experiments using was performed. As a result, after the correction, the CV value between 10 measurements experiments expression level of each miRNA, the median for the entire approximately 1000 types of miRNA detected, respectively 0.32 (SEQ ID NO: 1), 0.40 (sequence number 2), 0.40 (SEQ ID NO: 3), 0.38 (SEQ ID NO: 4), 0.30 (SEQ ID NO: 5), 0.41 (SEQ ID NO: 15), 0.36 (SEQ ID NO: 16), was 0.39 (SEQ ID NO: 17).

[0195]

　as a standard, instead of the standard material for the embodiment shown in SEQ ID NO: 1-5, using standard nine comparative example shown in SEQ ID NO: 6-14, Example 1 a similar experiment was carried out with.

[0196]

　Before correction, the center value of the detected approximately 1000 types of expression level of the CV value of each miRNA was 0.45.

[0197]

　After the correction, the median expression level of the CV value of each miRNA are respectively 1.07 (SEQ ID NO: 6), 1.14 (SEQ ID NO: 7), 0.98 (SEQ ID NO: 8), 0.99 (SEQ ID NO: 9), 0.94 (SEQ ID NO: 10), 0.95 (SEQ ID NO: 11), 0.84 (SEQ ID NO: 12), 0.82 (SEQ ID NO: 13), was 0.88 (SEQ ID NO: 14).

[0198]

　As described above, while the median CV value of the detection values ​​of target small RNA after correction in the first embodiment is smaller than 0.45 before correction, it is greater than 0.45 before correction in Comparative Example 1. That is, the correction of the same specimen expression level of variation between the experiments in multiple experiments performed under the same conditions to DNA microarray experiments from the extraction of nucleic acids using (variation), compared to Comparative Example 1, by performing correction using the standard used in example 1, the accuracy of the correction is shown to be improved.

[0199]

　using three kinds of serum A ~ C taken from three The target size RNA (miRNA), similarly as in Example 1, using about 1000 types of miRNA detected.

[0200]

　Correction of the measured values ​​of each miRNA in the target small RNA was performed as follows.

[0201]

　First, the abundance of each standard material obtained in the measuring step, is calculated as the mean value of the measured values ​​of the eight points on the DNA microarray, which bottom is converted to 2 logarithmic values. Then, an average value of the log-transformed values ​​of the five standard material obtained was a representative value of this. As described above, for each serum A ~ C, it was calculated first and second representative value. Logarithmic conversion value and the average value thereof for each standard (the representative value) shown in Table 2.

[0202]

　Correction of the expression level of each miRNA between two experiments serum A ~ C are, the first representative value as the reference value, the difference between the second representative value (-1 th second), in each serum and the correction coefficient (Table 3).

[0203]

　Then, the measured value of expression amount of miRNA in the serum, each bottom into a second logarithmic, was performed the correction from the second logarithmic conversion value of each serum by subtracting a correction coefficient in each serum of the .
　By the above operation, the correction of the measured values of the second miRNA expression level of experiments were performed.

[0204]

　using a standard material for the embodiment shown in SEQ ID NO: 15-17 as the standard, was carried out the same experiment as in Example 2.

[0205]

　using a standard material for comparative example shown in SEQ ID NO: 6-9 as a standard, was carried out the same experiment as in Example 2.

[0206]

　Using the standard for comparison example shown in SEQ ID NO: 10-14 as the standard, was carried out the same experiment as in Example 2.

[0207]

　Example 2, Example 3, Comparative Example 2, the correction expression of miRNA for the first time for each experiment of Comparative Example 3 measurements of the expression level of a target small RNA (reference sample) and second (analyte to be corrected) the results of calculating the regression line of the measured values ​​of the amount shown in Table 4, also, the first horizontal axis (reference analyte, no correction), second on the vertical axis (the analyte to be corrected, correction is made) plot the expression level of the scatter plot figure (serum a) 4 ((a): result of the correction of example 2, (B): the results of the correction of Comparative example 2) are shown in.

[0208]

　Because of experiments first and twice using the same serum, originally both results (1st 2nd results of the samples of which receives the results and the correction of the analyte) is should coincide, the If, regression line should overlap on the line of y = x. Comparing Comparative Examples 2 and 3 and Examples 2 and 3, the slope was the same both. However, embodiment the absolute value of the intercept at 2,3 is 0.5, It deviates significantly from a straight line y = x.

[0209]

　In above manner, Examples 2 and 3, between the two measurement experiment, the absolute value of the intercept of the regression line correction data are obtained close to y = x 0, the accuracy of the data after correction it can be said that has been shown to be high. On the other hand, in Comparative Examples 2 and 3, the absolute value of the intercept of the regression line is larger than 0.5, it would be less accurate data after correction is shown.

[0210]

[Table 2]

[0211]

[table 3]

[0212]

[Table 4]

[0213]

　As confirmed in Examples 1 to 3 and Comparative Examples 1 to 3 described above, by using the correction method of the present invention, accurate data for comparative analysis of the expression level of a target small RNA contained in a plurality of analytes It can be obtained, allowing accurate analysis of expression levels of the target small RNA.

DESCRIPTION OF SYMBOLS

[0214]

10 analyzer
110 input unit
120 display unit
130 output unit
140 storage unit
150 control unit
160 conversion unit
170 analyzer

The scope of the claims

[Claim 1]

　A method of correcting the expression level for comparative analysis of the expression level of a target small RNA in a plurality of analytes,
　said plurality of each specimen, at least one standard nucleic acid length of 200 bases or more nucleic acid added and then, extraction step extracts nucleic acid from the sample to obtain a nucleic acid sample;
　the amount of target small RNA and standards present in each nucleic acid sample extracted was measured respectively, the expression level of the target small RNA for each sample and obtaining a measure of the amount of extracted standard, measuring step;
　for each sample, to obtain a representative value from the measured value of the amount of extracted standard, the representative value acquiring step;
　is arbitrarily set with respect to the extracted amount of standard a reference value, the Samata the ratio of representative values of the standard of each sample obtained by the representative value acquiring step acquires as the correction coefficient of the target small RNA expression levels for each sample, the correction coefficient acquiring step;
　obtaining each correction coefficient that is it Used, the correction step of correcting the expression level of the measured target small RNA for each sample;
including, correction method.

[Claim 2]

　Correction method according to claim 1 nucleic acid length of the standard substance is 200 or more bases 1200 bases or less.

[Claim 3]

　At least one standard comprises at least one selected from standard is a nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1-5 and 15-17, the correction method of claim 2 wherein.

[Claim 4]

　Using two or more kinds of standards, the correction method according to any one of claims 1 to 3.

[Claim 5]

　Correction method according to any one of claims 1 to 4 specimen is a body fluid derived specimen.

[Claim 6]

　Correction method according to any one of claims 1 to 5 target small RNA is miRNA.

[Claim 7]

　The extraction of the nucleic acid sample in the extraction step is carried out by phenol-chloroform method, the correction method according to any one of claims 1 to 6.

[8.]

　Wherein the determining step, a labeled nucleic acid sample with a labeling substance, the probe is contacted with for capturing the probe and at least one standard for capturing the immobilized plurality of target small RNA on a support performs hybridization Te comprises obtaining an extract of the expression level and standard of each target small RNA as a signal strength measurement, the correction method according to any one of claims 1 to 7.

[Claim 9]

　Any representative value obtained by the representative value acquiring step, was calculated from the measured value of at least one standard extraction amount, an average value or median was represented by logarithm, of claims 1 to 8 correction method according to any one of claims.

[Claim 10]

　The reference value is fixed numbers are arbitrarily defined in terms extraction of standards, or are representative of the acquired standard extraction amount for the first analyte which is arbitrarily selected from the plurality of samples, wherein correction method according to any one of claims 1 to 9.

[Claim 11]

　Wherein the correction step,
(a) in the correction coefficient acquiring step, to acquire the value obtained by subtracting the reference value from the representative value as the correction coefficients, by subtracting the correction factor from the measured value of expression amount of the target small RNA ,
(b) in the correction coefficient acquiring step, to acquire the value obtained by subtracting the representative value from the reference value as a correction factor, adding the correction factor to the measured value of expression amount of the target small RNA,
(c) in the correction coefficient acquiring step, to acquire the value obtained by dividing the representative value with the reference value as a correction factor, it is divided by the correction factor the measured value of expression amount of the target small RNA, or
; (d) correction factor the acquisition step, when obtaining a value obtained by dividing the reference value in the representative value as the correction coefficient, multiplying the correction coefficient to the measured value of expression amount of the target small RNA,
by performing the correction, claims 1 10 of Zureka method described in (1).

[Claim 12]

　An apparatus for correcting the expression level to compare analyzing the expression level of a target small RNA in a plurality of specimens,
　in each of a plurality of analytes, at least one standard nucleic acid length of 200 bases or more nucleic acid added and then, it was measured using a nucleic acid sample obtained by extracting the nucleic acid from the sample, the storage means and for storing the measured values of the target small RNA expression levels and standards extraction amount for each sample;
　each sample for, obtains a representative value from the measured values of standard extraction amount, and the representative value acquiring unit;
　representatives of the arbitrarily set reference value with respect to the amount of extracted standard, each sample acquired by the representative value acquiring means the Samata the ratio of values, to obtain respectively a correction coefficient of the target small RNA expression levels for each sample, the correction coefficient acquiring means and;
　using each correction coefficient acquired by the correction coefficient acquiring section, the respective the specimen There correcting the expression level of the measured target small RNA, the correcting means
including said device.

[Claim 13]

　The representative value is calculated from the measured value of the at least one standard extraction amount, an average value or median was represented by logarithm, apparatus according to claim 12.

[Claim 14]

　The apparatus of claim 12 or 13 target small RNA is miRNA.

[Claim 15]

　Wherein the correction means,
in (a) the correction coefficient acquiring section, when acquiring a value obtained by subtracting the reference value from the representative value as the correction coefficients, by subtracting the correction factor from the measured value of expression amount of the target small RNA ,
(b) in the correction coefficient acquiring section, when acquiring a value obtained by subtracting the representative value from the reference value as a correction factor, adding the correction factor to the measured value of expression amount of the target small RNA,
(c) in the correction coefficient acquiring section, when acquiring a value obtained by dividing the representative value with the reference value as a correction factor, is divided by the correction factor the measured value of expression amount of the target small RNA, or
; (d) correction factor the acquisition unit, when obtaining a value obtained by dividing the reference value in the representative value as the correction coefficient, multiplying the correction coefficient to the measured value of expression amount of the target small RNA,
the correction performing, to claims 12 by 14 Noi Apparatus according to Re preceding paragraph.

[Claim 16]

　Stored in the storage means, the measurement value of the target expression amount of small RNA and standards extracted amount in a plurality of analytes, the labeled nucleic acid sample with a labeling substance, a plurality of targets small immobilized on a support RNA probe is contacted to capture the probe and at least one standard for capturing and subjected to hybridization, measuring respectively the amount of extracted expression levels and standards of each target small RNA as a signal strength measurement value is a value, according to any one of claims 12 to 15.

[Claim 17]

　To correct the expression level for comparative analysis of the expression level of a target small RNA among a plurality of analytes, one or more computers,
　a plurality of each specimen, at least a nucleic acid length of 200 bases or more nucleic acid after addition of one standard, using a nucleic acid sample obtained by extracting the nucleic acid from the sample, the amount of target small RNA and standards present in each nucleic acid sample was measured, each sample obtain a measure of the expression level of the target small RNA and extraction of standards for the measurement process;
　for each sample, to obtain a representative value from the measured value of the amount of extracted standard, the representative value acquiring step;
　extracting the standard a reference value which is arbitrarily set for the amount, the Samata the ratio of representative values of the standard of each sample obtained by the representative value acquiring step acquires as the correction coefficient of the target small RNA expression levels for each sample , the correction coefficient acquiring step; and
　Obtained it has been each correction coefficient using each correction step of correcting the expression level of the measured target small RNA for each sample
program for executing.

[Claim 18]

　To correct the expression level for comparative analysis of the expression level of a target small RNA among a plurality of analytes, one or more computers,
　a plurality of each specimen, at least the nucleic acid length is 200 bases more nucleic acid after addition of one standard, was measured using a nucleic acid sample obtained by extracting the nucleic acid from the sample, it stores the measurement value of the target small RNA expression levels or standard extraction amount for each sample ; storage means
　for each sample, to obtain a representative value from the measured values of standard extraction amount, the representative value acquiring means;
　and optionally set reference value with respect to the amount of extracted standard, acquired by the representative value acquiring means the Samata the ratio of the representative value of each sample were, respectively to obtain the correction coefficient acquiring unit as the correction coefficient of the target small RNA expression levels for each sample; and
　each correction coefficient acquired by the correction coefficient acquiring section There are, to correct the expression level of the measured target small RNA in each sample, the correction means
programmed to function as a.

[Claim 19]

　Recording a program according to claim 17 or 18, a computer readable recording medium.

[Claim 20]

　A probe for capturing a plurality of target small RNA, and a probe for capturing at least one standard selected from standard is a nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1-5 and 15-17 containing immobilized support, small RNA expression analysis chip.