Entitled: miRNA expression levels of comparative analysis methods and apparatus
Technical field
[0001] The present invention relates to a method and apparatus for comparative analysis of expression levels of miRNA included in the plurality of body fluid analytes.
Background technique
[0002] miRNA (micro RNA), the 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. Then, 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.
[0003] Recently, miRNA not only the cells, the cells more exist in body fluids, serum, plasma, urine, spinal fluid and the like is a sample that does not contain, the expression level of various diseases, including cancer Bio likely to be a marker has been suggested. 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.
[0004] On the other hand, in the case of gene expression analysis using a DNA microarray, the sample and experimenter, the experimental conditions, it is known that there is a possibility that an error in the resulting data may occur. Therefore, the data correction method for correcting errors have been devised.
[0005] The correction, which taken expression data of multiple genes as a by gene expression data set lump, the method the expression of the entire gene expression data set, that assumes the principle that there is no difference even if any specimen is It has been used. Global normalization method in this way, quantile method, lowess method, and the like 75 percentile method.
[0006] Further, paying attention to a particular gene expression level is identical between the specimens (such as beta-actin or GAPDH), as the detected value of the gene is constant, it has been performed a method of correcting the data for each sample .
[0007] As a method of correcting such that the expression level of a particular miRNA becomes constant among the non-coding RNA expressed in the sample, it is to be the expression level in various analyte is constant, housekeeping RNA (U1 snoRNA, U2 snoRNA, U3 snoRNA, U4 snoRNA, U5 snoRNA, U6 snoRNA, 5S rRNA, a method of correction using the 5.8S rRNA) has been proposed (Patent documents 1 and 2). That is, Patent Document 1, Patent Document 2, by performing an operation such as detection values ​​of the 5S rRNA detected simultaneously with the detection of the miRNA is constant in all the samples, and correct the detection result of the miRNA.
[0008] On the other hand, since the DNA microarray to measure the number of miRNA simultaneously, a method of correction using only the limited number of housekeeping RNA is the lack of accuracy, also measuring the expression level of miRNA simultaneously a plurality of mRNA and a method of performing correction of miRNA using the expression level of the plurality of mRNA have been proposed (Patent Document 3). In Patent Document 3, by performing an operation such as detection values ​​of a plurality of mRNA detected simultaneously with the detection of miRNA is constant in all the samples, and correct the detection result of the miRNA.
[0009] In Non-Patent Document 1, in the measurement of miRNA expression levels in mouse serum using the endogenous miRNA to the correction of the expression level, and evaluate its effectiveness. Note that the endogenous miRNA, (in the case of Non-Patent Document 1 mouse serum) in a sample of interest indicates that the exist naturally, a miRNA derived from an organism that provided the sample. That is, in Non-Patent Document 1, by using the correction endogenous miR-16 as an example of a miRNA, miR-31, miR-223, but trying to correct the miRNA expression levels in mouse sera, these miRNA since the expression level was not constant between individuals, a method of performing the entire data corrected by the expression level of the endogenous miRNA is not employed, the use of the data correcting RNA added from outside as a reference substance It is proposed. In short, we conclude that it is not appropriate to the correction of the miRNA expression levels using these endogenous miRNA.
[0010] Non-Patent Document 2, when detecting a miRNA in human serum by quantitative RT-PCR and sequencing, let-7d, let-7g, and advocates the use as correction endogenous miRNA in combination the let-7i there. That, let-7d, let-7g, the expression level of the let-7i by performing an operation of the constant between specimens, are corrected expression level of miRNA to be measured.
CITATION
Patent Document
[0011] 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. 2014-7995

Non-patent literature
[0012] Non-Patent Document 1: Roberts, TC et al., 2014, PLoS ONE, 9 (2) winding, E89237
Non-Patent Document 2: Chen, X. et al., 2013, PLoS ONE, 8 (11) winding, E79652

Summary of the Invention
Problems that the Invention is to Solve
[0013] As described above, when detecting the gene expression, using the expression level of a small variation in the expression level between sample "housekeeping genes", a method of correcting the expression level of the gene to be detected is generally used ing. The housekeeping genes that are particularly well-known, ACTB, such as GAPDH, and the like. However, these genes, miRNA and are significantly different absolute values ​​of the nucleotide length or expression amount, be used for the purpose of correcting the expression level of miRNA is not preferable.
[0014] Therefore, conventionally, in the case of detecting the expression level of miRNA is used for U1 snoRNA, U2 snoRNA, U3 snoRNA, U4 snoRNA, U5 snoRNA, U6 snoRNA, 5S rRNA, housekeeping RNA such 5.8S rRNA is mainly corrected It is to have. However, measuring the expression level of miRNA in a body fluid, if you try to correct the data, U1 snoRNA, U2 snoRNA, U3 snoRNA, U4 snoRNA, U5 snoRNA, U6 snoRNA, 5S rRNA, 5.8S rRNA is the nucleus since a RNA present in or cytoplasm, can not be detected serum, plasma, urine, little expression level in bodily fluids of spinal fluid such as a sample containing no cells, expression of a miRNA of the body fluid it is that it can not be an indicator for the amount of detection and correction.
[0015] In the expression level detected in the miRNA by quantitative RT-PCR method, the miRNA present in the serum or plasma by analyzing separately, try to find a "body fluid housekeeping miRNA" is not the expression level difference between the sample an attempt has been carried out. As described above, Non-Patent Document 2, when detecting a miRNA in human serum by quantitative RT-PCR and sequencing, let-7d, let-7g, advocates the use in combination of the let-7i. However, whether these miRNA functions as correction endogenous miRNA even DNA microarray method is unknown. That is, in the miRNA analysis in a body fluid using a DNA microarray was no effective correction method using the specific correction endogenous miRNA.
[0016] On the other hand, when carrying out the correction of the measured values ​​in the DNA microarrays, line it is generally corrected by using the total amount of gene expression of many measurable (several hundreds to several tens of thousands of) (signal value) in DNA microarrays are we. For example, global normalization method, quantile normalization method, etc., are mentioned as the method.
[0017] Incidentally, when an index of a reference value obtained from the expression level of miRNA, if a number miRNA as a source data for forming the reference value, there is a problem that whether can form a universal correction reference value . In particular, the number of miRNA to be measured is limited to several to several tens, in the DNA microarray application specific, applying and global normalization method, is likely to result in poor compensation results. Also, by measuring the expression level of a large number of mRNA in a sample, which method of Patent Document 3, the reference value for correction is effective for tissue or cell-derived RNA, mRNA in body fluids the amount of expression is very low, it is difficult to them as an index for the expression level correction of miRNA.
[0018] As described above, measuring the expression level of miRNA included in the plurality of body fluid analytes, when attempting to comparative analysis, it has not been found an effective method for correcting the measured value. In particular, the method can be performed valid correction using a small number of correction endogenous miRNA by DNA microarray does not exist. The present invention is intended to solve the problems as described above, it is intended to particularly solve critical issues in practical application of a relatively small number of miRNA measuring DNA microarrays, such as testing and diagnostic applications .
Means for Solving the Problems
[0019] The present inventors have found, after intensive studies to solve the above problems, and completed the following invention.
(1) A method of comparative analysis of the expression level of a target miRNA across multiple bodily fluid analyte,
the plurality of body fluid analytes, from the correction endogenous miRNA shown in measurement and SEQ ID NO: 1-10 in the expression level of the target miRNA performing one or more of the measurement of the expression amount of the correction endogenous miRNA simultaneously selected, the measuring step;
for each body fluid sample, one or more of correction is selected from the correction endogenous miRNA shown in SEQ ID NO: 1-10 obtaining a representative value from the measured value of expression amount of use endogenous miRNA, representative value acquiring step;
and optionally set reference values for the expression amount of the correction endogenous miRNA, each body fluid obtained by the representative value acquiring step the Samata the ratio of the value representative of the analyte, respectively acquires a correction coefficient of the target miRNA expression level for each body fluid sample, the correction coefficient acquiring step; and
using respectively the acquired correction coefficient for each body fluid sample , measured in each body fluid sample By correcting the expression level of the target miRNA was, correction step
including the method.
(2) In the correction step,
(a) the in the correction coefficient acquiring step, to acquire the value obtained by subtracting the reference value from the representative value as the correction coefficient, subtracts the correction factor from the measured value of expression amount of the target miRNA to it,
(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 miRNA,
(c ) wherein in the correction coefficient acquiring step, to acquire the 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 miRNA, 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 miRNA,
by performing the correction, according to (1) the method of.
(3) the reference value, a fixed numerical value which is arbitrarily set with respect to the correction endogenous miRNA expression level, or the first correction endogenous obtained for body fluid specimen is arbitrarily selected from the plurality of bodily fluid analyte are representative of miRNA expression levels, the method described in (1) or (2).
(4) the measurement process, one or a plurality of correction are selected from the correction endogenous miRNA shown in the nucleic acid probe and SEQ ID NO: 1-10 for capturing a plurality of target miRNA immobilized on a support a probe for capturing an endogenous miRNA, a nucleic acid sample derived from the labeled body fluid sample contacted to a labeling substance subjected to hybridization, the expression level of the target miRNA and the one or more correction endogenous miRNA each includes obtaining a signal strength measurement method according to any one of (1) to (3).
(5) the body fluid sample is blood, serum or plasma, the method according to any one of (1) to (4).
Mean (6) the representative value is calculated from the measured values of one or more correction endogenous miRNA expression levels are selected from the correction endogenous miRNA shown in SEQ ID NO: 1-10, represented by logarithm a value or median a method according to any one of (1) to (5).
(7) The miRNA expression analysis device for comparative analysis of the expression level of a target miRNA in a plurality of body fluid analytes,
measured for a plurality of bodily fluid analyte, shown in measurements and SEQ ID NO: 1-10 in the expression level of the target miRNA storage means for storing the measured value of expression amount of one or more correction endogenous miRNA is selected from the correction endogenous miRNA;
for each body fluid sample is selected from the correction endogenous miRNA shown in SEQ ID NO: 1-10 obtaining one or more of the representative value from the measured value of expression amount of the correction endogenous miRNA that, the representative value acquiring means;
and optionally set reference values for the expression amount of the correction endogenous miRNA, the representative value acquiring the Samata the ratio of the representative value of each body fluid sample obtained by means acquires respectively as the correction coefficient of the target miRNA expression level for each body fluid sample, the correction coefficient acquiring section;
acquired for each body fluid sample each of the correction coefficient There are, for correcting the expression level of the measured target miRNA in the body fluid sample, the correction means; and
the corrected target miRNA expression levels, and outputs the result of comparing the target miRNA expression level between at least two fluid sample output means
including said device.
(8) said correction means,
(a) in the correction coefficient acquiring section, when acquiring a value obtained by subtracting the reference value from the representative value as the correction coefficient, subtracts the correction factor from the measured value of expression amount of the target miRNA to it,
(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 miRNA,
(c ) wherein the correction coefficient acquiring section, when acquiring a 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 miRNA, 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 miRNA,
performs the correction by, according to (7) device.
(9) wherein is stored in the storage means, one or a plurality of correction are selected from the correction endogenous miRNA shown in measurements and SEQ ID NO: 1-10 in the expression level of the target miRNA contained in each of a plurality of bodily fluid analyte measurement of the expression level of endogenous miRNA is a probe for capturing the probe and said one or more correction endogenous miRNA for capturing a plurality of target miRNA immobilized on a support, a labeling substance in a nucleic acid sample derived from the labeled body fluid sample is contacted performs hybridization, a value measured respectively the expression level of the target miRNA and the one or more correction endogenous miRNA as signal strength measurements, apparatus according to (7) or (8).
(10) for comparing analyzing the expression level of a target miRNA across multiple bodily fluid analyte, one or more computers,
for each of a plurality of bodily fluid analyte, shown in the measurement and SEQ ID NO: 1-10 in the expression level of the target miRNA to measure the expression level of one or more correction endogenous miRNA is selected from the correction endogenous miRNA simultaneously measuring step;
for each body fluid sample is selected from the correction endogenous miRNA shown in SEQ ID NO: 1-10 obtaining a representative value 1 or a plurality of the measurement of the expression amount of the correction endogenous miRNA that, the representative value acquiring step;
and optionally set reference values for the expression amount of the correction endogenous miRNA, representative value acquiring Samata the representative value of each body fluid sample obtained in step a ratio obtains respectively as the correction coefficient of the target miRNA expression level for each body fluid sample, the correction coefficient acquiring step; and
acquired for each body fluid sample each of the correction coefficient Used to correct the expression level of the measured target miRNA in the body fluid sample, the correction process
program for executing.
(11) for comparing analyzing the expression level of a target miRNA across multiple bodily fluid analyte, one or a plurality of computers,
measurements and SEQ ID NO: 1-10 in the expression level of the target miRNA contained in each of a plurality of bodily fluid analyte storage means for storing the measured value of expression amount of one or more correction endogenous miRNA is selected from the correction endogenous miRNA shown in;
for each fluid sample, from the correction endogenous miRNA shown in SEQ ID NO: 1-10 obtaining a representative value from the measured value of expression amount of one or more correction endogenous miRNA is selected, the representative value acquiring means;
and optionally set reference values for the expression amount of the correction endogenous miRNA, the representative the Samata the ratio of the representative value of each body fluid sample obtained by the value obtaining means obtains respectively as the correction coefficient of the target miRNA expression level for each body fluid sample, the correction coefficient acquiring section;
acquired for each body fluid sample a correction coefficient that is it With which to correct the expression level of the measured target miRNA in the body fluid sample, the correction means; and
the corrected target miRNA expression levels, the results of comparing the target miRNA expression level between at least two fluid sample output means for outputting
program for functioning as a.
(12) (10) or (11) recording a program described in a computer-readable recording medium.
(13) one or more probes to capture correction endogenous miRNA is selected from the correction endogenous miRNA shown in the probe and SEQ ID NO: 1-10 for capturing a plurality of target miRNA is immobilized comprising a support, chip miRNA expression analysis.
Effect of the Invention
[0020] According to the present invention, when comparing analyzing the expression level of a target miRNA included in the plurality of body fluid analytes, especially measuring the expression level of a large number of miRNA using microarray or the like, when comparing miRNA expression levels between the specimen the can than conventional accurately correct the miRNA expression levels. The present invention, comparative analysis of the target miRNA between analyte will be able to practice more accurately.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] 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 expression level of a target miRNA in accordance with the present invention.
[Figure 4-1] measured by the method shown in Example 2 shows a histogram of the miRNA signal values by the corrected DNA microarray detection. A: before correction, B: correction by hsa-miR-6085, C: correction by hsa-miR-1227-5p, D: correction by hsa-miR-2861, E: correction by hsa-miR-149-3p, F : correction by hsa-miR-4463, G: correction by hsa-miR-4508
measured by the method shown in [Figure 4-2] example 2 shows a histogram of the miRNA signal values by the corrected DNA microarray detection. A: before correction, H: Correction by hsa-miR-6090, I: correction by hsa-miR-6775-5p, J: correction by hsa-miR-6803-5p, K: correction by hsa-miR-5787
[FIG. 5] measured by the method shown in example 2 shows a histogram of the miRNA signal values by the corrected DNA microarray detection.
[6] measured by the method shown in Comparative Example 1 shows a histogram of the miRNA signal values by the corrected DNA microarray detection.
[7] measured by the method shown in Comparative Example 2 shows a histogram of the miRNA signal values by the corrected DNA microarray detection.
[Figure 8-1] measured by the method shown in Example 4 shows a histogram of the miRNA signal values by the corrected DNA microarray detection. A: before correction, B: hsa-miR-149-3p , hsa-miR-1227-5p, corrected by the combination of hsa-miR-2861, C: hsa-miR-149-3p, hsa-miR-1227-5p , corrected by the combination of hsa-miR-4508, D: hsa-miR-149-3p, hsa-miR-1227-5p, corrected by the combination of hsa-miR-4463, E: hsa-miR-149-3p, hsa -miR-2861, corrected by the combination of hsa-miR-4508
measured by the method shown in FIG. 8-2] example 4 shows a histogram of the miRNA signal values by the corrected DNA microarray detection. A: before correction, F: hsa-miR-149-3p , corrected by the combination of hsa-miR-2861, hsa- miR-4463, G: hsa-miR-1227-5p, hsa-miR-2861, hsa-miR correction of a combination of -4508, H: hsa-miR- 1227-5p, corrected by the combination of hsa-miR-2861, hsa- miR-4463, I: hsa-miR-1227-5p, hsa-miR-4508, hsa correction by the combination of -miR-4463

DESCRIPTION OF THE INVENTION
[0022] First, the concept of a correction method of a target miRNA performed in the present invention will be described with reference to FIG. In Figure 1, labeled RNA in a body fluid sample is selected from the probes to capture a plurality of types of target miRNA (hereinafter, also referred to as "target miRNA capture probe".) And miRNA shown in SEQ ID NO: 1-10 1 or more miRNA (hereinafter, also referred to as "correction endogenous miRNA.") probes to capture (hereinafter, referred to as "correction endogenous miRNA capture probe".) the encounters a fixed microarray results the schematically shows the histograms of the signal values ​​is a measure of the expression level of each miRNA. Hereinafter, a probe for capturing a probe or compensating endogenous miRNA for capturing a target miRNA, also collectively called "miRNA capture probe" or simply "probe".
[0023] In Figure 1A, the target miRNA in the sample extracted from a bodily fluid specimen A and bodily fluid analyte B, and results of each analyzed using DNA microarrays are indicated by the histogram. Obtained signal values ​​derived from a plurality of target miRNA capture probe mounted on the microarray, and shows a histogram of the signal values ​​obtained from the correction endogenous miRNA capture probe of the present invention, respectively. In bodily fluid analyte A body fluid sample B, miRNA histogram is deviated. Therefore, it can be interpreted that there is a large difference in the expression level of miRNA between specimens. On the other hand, it can be interpreted as the difference is caused by the experimental error. Which is correct, it can not be determined only from the histogram of the miRNA.
[0024] Meanwhile, if here there are correction endogenous miRNA abundance in body fluids is always constant, the value of the signal value of the correction for endogenous miRNA should be consistent between samples.
[0025] Shown in FIG. 1A, a histogram of the signal values ​​obtained from the correction endogenous miRNA capture probe indicates almost the same distribution in the body fluid sample A body fluid sample B. In other words, bodily fluid analyte A body fluid 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 miRNA between bodily fluid analyte AB, the correction of the signal value of the miRNA when the comparisons between the body fluid sample is not required.
[0026] 1B, the are the result of analyzing the body fluid sample C and bodily fluid analyte D using a DNA microarray is schematically shown. Signal values ​​obtained from the target miRNA capture probe, and respectively show the signal value obtained from the correction endogenous miRNA capture probe.
[0027] In the body fluid sample C and bodily fluid analyte D, miRNA histogram indicates a similar distribution. On the other hand, histograms of the signal values ​​obtained from the correction endogenous miRNA capture probe is significantly deviated in a body fluid sample C and bodily fluid analyte D. The detection result of the body fluid sample C and bodily fluid analyte D This indicates that experimental error is caused by some reason. Upon the comparison between bodily fluid analyte CD in such cases, it is necessary to appropriately correct the signal value of the miRNA.
[0028] In accordance with the present invention, showing a histogram after correction of the signal value of the target miRNA in Figure 1C. Specific methods of correction is described as follows. So that the histogram of the signal values ​​obtained from the correction endogenous miRNA capture probe body fluid sample C and bodily fluid analyte D match, data were corrected for body fluid specimen C. By this correction, the correction histogram of the signal values ​​obtained from the endogenous miRNA capture probe comes to coincide with the body fluid sample C and bodily fluid analyte D, corrected signal value of the target miRNA capture probe using the same correction factor of the histogram, come largely shifted. In other words, even between specimens CD, it comes to a large difference is the expression of the target miRNA.
[0029] Here is shown a method for correcting the signal values ​​of miRNA between two body fluid analytes, bodily fluid analyte to compare is not limited to two, it may be compared indefinitely. When comparing three or more body fluid samples, for example, the advance correction for endogenous miRNA capture signal value obtained from the probe, previously assuming a constant number (constant), for the constant of the body fluid sample by calculating the difference or ratio of the signal values ​​obtained from the correction endogenous miRNA capture probe, either adds or subtracts the difference to the signal value of the target miRNA in the body fluid sample, the reciprocal of the ratio in the body fluid sample by multiplying or division on the signal value of the target miRNA, it can be easily performed multiple correction between specimens.
[0030] In the present invention, comparative analysis of the expression levels of miRNA between multiple analytes. Specimen number may be two, or may be three or more.
[0031] The term "miRNA" is a type of non-coding RNA (ncRNA), which refers to a chain length of 15 to 25 bases about the short RNA produced in vivo, it is believed to have the ability to modulate the expression of mRNA . 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. Then, 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 is its presence has been confirmed in a variety of organisms.
[0032] Analyte to which the present invention can be applied is a body fluid sample separated from a living body, such as blood, serum, plasma, urine, spinal fluid, saliva, swabs, there may be mentioned body fluids of various tissue fluid or the like, these the present invention is not limited. A plurality of analyte comparative analysis may be a plurality of analytes from different body fluids, may be a plurality of body fluid specimens from the same type of body fluid isolated from a different organism.
[0033] RNA was extracted from these specimens, measuring the expression level of miRNA using the RNA. Method for extracting such RNA are known (e.g., Favaloro et al method (Favaloro et.al., Methods Enzymol.65: 718-749 (1980)), etc.), have also been various commercial kits for its ( for example, Qiagen of miRNeasy, of Toray Industries, Inc. "3D-Gene" RNA extraction reagent from liquid sample, etc.).
[0034] Referred to as "endogenous" in the present invention, artificially rather than being added to the sample, which means that naturally present in the sample. For example, when said "endogenous miRNA" refers to a miRNA derived from that naturally present in the sample, it provided the sample organism.
[0035]
In the comparative analysis method of the present invention, simultaneously with the measurement of the expression level of a plurality of types of target miRNA in the sample, also performs the measurement of one or more of the expression amount of the correction endogenous miRNA. Expression level of the correction endogenous miRNA, as described below, is used to calculate the correction coefficient for correcting the expression level of the target miRNA. Simultaneous measurement of the expression level of the plurality of miRNA is, for example, can be performed probe that specifically binds to the miRNA target was immobilized on a support, by hybridization assays using array chip microarray, or the like. In the present invention, a plurality of target miRNA capture probe and one or more correction endogenous miRNA capture probe may be used array chip containing immobilized support.
[0036] The "capture probe" or "probe for capturing", directly or indirectly, and miRNA capturing object, preferably directly, and means a substance capable of selectively binding, as a typical example it can include nucleic acids, proteins, sugars and other antigenic compounds. In the present invention, it can be preferably used a nucleic acid probe. Nucleic acid, in addition to DNA and RNA, it is possible to use a nucleic acid derivative such as PNA (peptide nucleic acid), or LNA (Locked Nucleic Acid). Here, the derivative, in the case of nucleic acid, 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 a base re configuration, the double bonds of the saturated, deamination, means a chemically modified derivatives such as derivatives containing nucleotides, etc.) that received such substitution of sulfur molecules oxygen molecules.
[0037] The chain length of the nucleic acid probe in order to ensure the stability and specificity of hybridization, it is preferably not less than the length of the miRNA to be detected. Usually, 17 if ~ 25 bases about the chain length, the probe can be sufficiently exhibited selective binding to miRNA of interest. Such chain length short oligo nucleic acid probes can be readily prepared by well known chemical synthesis method.
[0038] Nucleic acid probe, it is preferable that a completely complementary nucleotide sequence and miRNA of interest, even if there are differences in some, high homology to the extent capable of hybridizing with miRNA stringent conditions of the target base if sequences can be used as capture probes.
[0039] 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 (1998) Molecular Cloning:. (. 2nd ed) A Laboratory Manual, or the like can be used conditions described in Cold Spring Harbor Laboratory Press, New York . 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 for obtaining the function as capture probes were prepared for detection of the desired analyte RNA.
[0040] Knowledge of the miRNA sequences 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/). Correction endogenous miRNA capture probe and the target miRNA capture probe can be designed based on sequence information available from those sites.
[0041] The number of miRNA capture probe that is immobilized on a support is not particularly limited. For example, the number to cover all known miRNA that sequences have been identified miRNA capture probe used in which immobilized on a support, may measure the expression levels of miRNA, desired number of the miRNA capture probe may be used as immobilized on a support.
[0042] The support capture probe is aligned immobilized, it is possible to use the same support that is used in known microarray and microarray or the like, it can be used, for example slide glass, membranes, beads, etc. . 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] Hybridization of each miRNA capture probe which is immobilized on a support, from RNA extracted from bodily fluid analyte, the labeled nucleic acid sample (nucleic acid sample from a body fluid sample) was prepared with a labeling substance, the labeled nucleic acid sample the carried out by contacting the probe. The "body fluid sample derived from a nucleic acid sample", in addition to the RNA extracted from the bodily fluid analyte, cDNA and cRNA prepared by reverse transcription reaction from the RNA and the like. The nucleic acid sample derived from the labeled body fluid analyte, may be one labeled analyte RNA directly or indirectly labeled substance, also, directly or indirectly labeled substance cDNA or cRNA prepared from the sample RNA in may be the one that was labeled.
[0047] As a method for binding a labeling substance to the nucleic acid sample from a body fluid specimen, '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 nucleotide labeled substance is bound to the nucleic acid and a method of incorporating. 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, 3 as a 'kit to bind directly or indirectly labeled substance to the end, "3D-Gene" miRNA labeling kit (Toray), miRCURY miRNA HyPower labeling kit (Ekishikon Co.), NCode miRNA Labeling system (Life Technologies Inc.) can be exemplified FlashTag Biotin RNA Labeling Kit (Genis Sphere Inc.).
[0048] In addition, as in the conventional method, by synthesizing the cDNA or cRNA from the specimen RNA in the presence of the labeled deoxyribonucleotide or labeled ribonucleotide, a cDNA or cRNA labeled substance was incorporated was prepared, which array to probe hybridized with the above, the method can also be called.
[0049] In the present invention, uses a plurality of analytes may be used the same labeling substance to both.
[0050] In the present invention, the labeling substance can be used, mention may be made of various labeling substances it is 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.
[0051] 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).
[0052] As described above by contacting a nucleic acid sample from a labeled bodily fluid analyte and miRNA capture probe on the support, thereby hybridizing the nucleic acid sample and a probe. 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.
[0053] Detected signal value is compared with the ambient noise. Specifically, the signal values ​​obtained from the probe immobilization region, and comparing the signal values ​​obtained from other positions, and detected a case where the former numerical values ​​are exceeded (valid determination positive) .
[0054] The detected signal values, if included background noise may be subtracted background noise. The ambient noise as the background noise can be subtracted from the detected signal values. Other, "Fujifukashiko, Katsuhisa Horimoto ed., Microarray data statistical analysis protocols, YODOSHA, 2008" may be using the method described in.
[0055] According to the above method, the measured value of expression amount of the correction endogenous miRNA and the target miRNA are obtained as a measure of signal strength.
[0056]
In the analysis method of the present invention, then, for each sample, representative values from the measured values of the expression amount of the correction endogenous miRNA, preferably obtains a representative value expressed in logarithmic value (representative value acquisition process). The "logarithmic value" in the present invention, the bottom means a value converted to 2 logarithm.
[0057] Average when using the plurality of correction endogenous miRNA, the representative value, an average or median calculated from each measurement of the expression amount of the plurality of correction endogenous miRNA, which is preferably represented by logarithm value or the center value is adopted. When using the correction endogenous miRNA only one measurement of the expression amount of the correction endogenous miRNA, it may preferably be employed as it is as the representative value is the logarithm of the measured value. Alternatively, as described later, when a plurality of capture probes with respect to one type of correction endogenous miRNA is using an array chips are spots, from the signal measurements from the plurality of spots (probe immobilization region) , the average or median, preferably an average value or median was represented by logarithm, it is a representative value.
[0058] The "average value expressed in logarithmic value", the measured value of expression amount of a plurality of correction endogenous miRNA (e.g., a microarray measurements of signal intensities obtained using) converted to the bottom 2 logarithm It means the average value obtained in the logarithmic value. If the representative value is the median, the "central value expressed in logarithmic value", the measured value of expression amount of a plurality of correction endogenous miRNA (e.g., measured values ​​of the signal intensities obtained using microarrays) the bottom median logarithm converted into 2 logarithm, or bottom of the median of the correction endogenous miRNA expression level measurements means logarithm converted into 2 logarithm. For median, it is subjected to logarithmic conversion of the measured value after be performed first the same value is obtained.
[0059] Mean and median, there is obtained with practically all of the measurements of the plurality of correction endogenous miRNA that was measured may be extracted from among the correction endogenous miRNA said plurality of been some measurements may be those obtained by using the. For example, it may be determined using the measurements of all the correction endogenous miRNA obtained in correction endogenous miRNA capture probe mounted on a microarray, one out of all the correction endogenous miRNA capture probe Part (for example, when the correction endogenous miRNA capture probe mounted on the microarray and was 10 species, three of which) may be obtained by extracting. Further, a plurality spots against one correction endogenous miRNA, may be a measurement value obtained from the correction endogenous miRNA capture probe. For example, it is possible to extract only the correction endogenous miRNA capture probe immobilization region was effective determination positive common to all specimens should be comparative analysis to obtain a representative value of the correction endogenous miRNA.
[0060] Then, the representative value of the correction endogenous miRNA of each sample obtained by the representative value acquiring step, using the arbitrarily set reference values ​​for the expression amount of the correction endogenous miRNA, of the target miRNA expression levels of obtaining a correction coefficient used for correction (correction coefficient acquisition step). The correction coefficient acquiring step can employ the steps of the correction coefficient acquiring step 1 or the correction coefficient acquisition step-2 below.
[0061]
correction coefficient acquisition step -1 is a method of utilizing a difference between the representative value and the reference value of the correction endogenous miRNA. In this step, the following 1-1. Reference analyte acquisition method, or 1-2. It may apply a fixed value correcting method.
[0062] 1-1. Reference analyte acquisition method
arbitrarily selects one sample (first sample) from a plurality of bodily fluid analyte to be detected in the miRNA, it is the "reference sample". The remaining one or more analytes (analyte second later), the "analyte to be corrected."
[0063] 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.
[0064] In this method, a representative value of the correction endogenous miRNA reference analyte and "reference value". And the reference value, the difference between the representative value of the correction endogenous miRNA of each sample 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.
[0065] Specifically, the correction coefficient is calculated by Equation 1 or 1 '.
[0066] c 1-1 = (representative value of the correction endogenous miRNA reference analyte (reference value))
- (a representative value of the correction endogenous miRNA of analyte to be corrected) Equation 1
c 1-1 '= ( representative value of the correction endogenous miRNA corrected the analyte)
- (representative value of the correction endogenous miRNA reference analyte (reference value)) equation 1 '
[0067] For example, measurements were performed of the expression amount using the microarray, the case of using the average value as a representative value of the correction endogenous miRNA, correction coefficients for the analyte to be corrected, be determined by the formula 2 or formula 2 ' it can.
[0068] [Number 1]

[0069] [Number 2]

[0070] Here, in Equation 2, Equation 2 ',
n is the total number of probe immobilization region for capturing the correction endogenous miRNA on the support,
Aj is the reference analyte, j-th (1 ≦ j ≦ n signal measurements from the probe immobilization regions to capture the correction endogenous miRNA in),
Xj is the second analyte, j-th (1 ≦ j ≦ n) for capturing the correction endogenous miRNA signal measurements from the probe immobilization regions,
a.
[0071] When the probe is corrected for endogenous miRNA one-to-one correspondence, n is a probe for capturing the correction endogenous miRNA on the support it is equal to the number of correction endogenous miRNA that target.
[0072] 'In, instead of n, the total number n of probe immobilization region for capturing the correction endogenous miRNA that was in effect determined positive common to all the samples to be compared' Formula 2 and Formula 2 using it is also possible.
[0073] 1-2. Fixed value correcting method
for a representative value of the correction endogenous miRNA, a pre-assumed way as to take a certain numerical in all specimens. That is, the numerical value fixed, with the difference between the representative value of the correction endogenous miRNA from each specimen, utilizing this difference as a correction coefficient. In this method, using the numerical values this fixed as "reference value". Correction factor will be acquired by the number of analytes to be corrected. It should be noted that, in this case, 1-1. Since the "reference sample" is not present as shown in, all of the plurality of analytes to be detected of the miRNA, and "analyte to be corrected."
[0074] Specifically, the correction coefficient is calculated by Equation 3 or Equation 3 '.
[0075] r 1-2 = (fixed numeric value (reference value))
- (a representative value of the correction endogenous miRNA of analyte to be corrected) Equation 3
r 1-2 '= (correction endogenous analyte to be corrected representative value of the miRNA)
- (fixed number (reference value)) equation 3 '
[0076] For example, when using the average value as a representative value of the correction endogenous miRNA, correction coefficients for the analyte to be corrected, can be obtained by equation 4 or equation 4 '.
[0077] [Number 3]

[0078] [Formula 4]

[0079] Here, Equation 4, wherein 4 ',
alpha is the reference value,
n is the total number of probe immobilization region for capturing the correction endogenous miRNA on the support,
Yj is the analyte, j-th (1 signal measurements from the probe immobilization regions to capture the correction endogenous miRNA of ≦ j ≦ n),
it is.
[0080] When the probe is corrected for endogenous miRNA one-to-one correspondence, n is a probe for capturing the correction endogenous miRNA on the support it is equal to the number of correction endogenous miRNA that target.
[0081] 'In, instead of n, the total number n of probe immobilization region for capturing the correction endogenous miRNA that was in effect determined positive common to all the samples to be compared' Formula 2 and Formula 2 using it is also possible.
[0082] 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, if at all times be decided to use the same numbers as the fixed number, it is possible to comparative analysis even among body fluid specimen was subjected to measurement of the expression amount for another day. But are not limited to, fixed numbers, one or more correction endogenous miRNA used for the acquisition of the correction coefficient can adopt a number of generally can take the expression level. Since the numerical value which can be taken to such common by a system used to measure the expression level may vary, the fixed numerical depending on the system to be used can be freely selected.
[0083] For example, 1 case of using the type of correction endogenous miRNA, an average value of the expression amount of the correction endogenous miRNA in a plurality of bodily fluid analyte comparison analyzed, may be used as a fixed value. When using the three types of correction endogenous miRNA, an average value of the miRNA expression of all of these three in a plurality of body fluid analytes, it may be used as a fixed value. Alternatively, it is acceptable to set a pre-fixed numerical values ​​using a number of bodily fluid analyte, it may be used repeatedly to the fixed number in subsequent analyzes.
[0084]
correction coefficient acquiring step-2 is a method of utilizing the ratio between the representative value and the reference value of the correction endogenous miRNA. In this step, the following 2-1. Reference analyte acquisition method, or 2-2. It may apply a fixed value correcting method.
[0085] 2-1. Reference analyte acquisition method
arbitrarily selects one sample (first sample) from a plurality of analytes to be detected in the miRNA, it is the "reference sample". The remaining second and subsequent sample, the "analyte to be corrected."
[0086] In this method, a representative value of the correction endogenous miRNA reference analyte and a "reference value", and the reference value, and the representative value of the correction endogenous miRNA of each sample in the second and subsequent (analyte to be corrected) the ratio of 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.
[0087] Specifically, the correction coefficient is calculated by Equation 5 or Equation 5 '.
[0088] c 2-1 = (representative value of the correction endogenous miRNA reference analyte (reference value)) /
(a representative value of the correction endogenous miRNA of analyte to be corrected) Equation 5
c 2-1 '= ( corrected the representative value of the correction endogenous miRNA analyte)
/ (a representative value of the correction endogenous miRNA reference analyte (reference value)) equation 5 '
[0089] For example, when performing the measurement of the expression amount using the microarray, using the average value as a representative value of the correction endogenous miRNA, the correction coefficient for the second analyte can be determined by Equation 6 or Equation 6 ' it can.
[0090] [Formula 5]

[0091] [Number 6]

[0092] Here, Formula 6, wherein 6 ',
n is the total number of probe immobilization region for capturing the correction endogenous miRNA on the support,
Aj is the reference analyte, j-th (1 ≦ j ≦ n signal measurements from the probe immobilization regions to capture the correction endogenous miRNA in),
Xj is the second analyte, j-th (1 ≦ j ≦ n) for capturing the correction endogenous miRNA signal measurements from the probe immobilization regions,
a.
[0093] When the probe is corrected for endogenous miRNA one-to-one correspondence, n is a probe for capturing the correction endogenous miRNA on the support it is equal to the number of correction endogenous miRNA that target.
[0094] 'In, instead of n, the total number n of probe immobilization region for capturing the correction endogenous miRNA that was in effect determined positive common to all the samples to be compared' Formula 6 and Formula 6 using it is also possible.
[0095] 2-2. Fixed value correcting method
for a representative value of the correction endogenous miRNA, a pre-assumed way as to take a certain numerical in all specimens. That is, for a fixed numeric value to obtain a ratio of the representative value of the correction endogenous miRNA from each specimen, utilizing this ratio as a correction coefficient. In this method, using the numerical values this fixed as "reference value". Correction factor will be acquired by the number of analytes to be corrected. It should be noted that, in this case, 2-1. Since the "reference sample" is not present as shown in, all of the plurality of analytes to be detected of the miRNA, and "analyte to be corrected."
[0096] Specifically, the correction factor is determined by Equation 7 or Equation 7 '.
[0097] r 2-2 = (fixed numeric value (reference value)) /
(a representative value of the correction endogenous miRNA of analyte to be corrected) Equation 7
r 2-2 '= (correction endogenous analyte to be corrected representative value of
MiRNA) / (fixed numerical value (reference value)) equation 7 '
[0098] For example, when using the average value as a representative value of the correction endogenous miRNA, correction coefficients for the analyte to be corrected can be calculated by Equation 8 or Formula 8 '.
[0099] [Number 7]

[0100] [Number 8]

[0101] Here, Formula 8, wherein 8 ',
alpha fixed numeric,
n is the total number of probe immobilization region for capturing the correction endogenous miRNA on the support,
Yj is the analyte, j-th (1 signal measurements from the probe immobilization regions to capture the correction endogenous miRNA of ≦ j ≦ n),
it is.
[0102] When the probe is corrected for endogenous miRNA one-to-one correspondence, n is a probe for capturing the correction endogenous miRNA on the support it is equal to the number of correction endogenous miRNA that target.
[0103] 'In, instead of n, the total number n of probe immobilization region for capturing the correction endogenous miRNA that was in effect determined positive common to all the samples to be compared' Equation 8 and Equation 8 using it is also possible.
[0104] As used herein, a reference value of the "fixed numerical" Details are similar to fixed numbers in "1-2. Fixed-value correcting method".
[0105] Then, it corrected using the obtained correction coefficient by the coefficient acquiring step 1 or the correction coefficient acquiring step-2, using the method of correction steps -1 or correction step-2, the expression of a target miRNA in the sample to be corrected the correction of the amount.
[0106]
correction process -1 is a method of correcting the expression level of the target miRNA by using a correction coefficient obtained by the correction coefficient acquiring step -1, adding a correction factor to the expression level of a target miRNA or performing corrected 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.
[0107] 1-1. Reference analyte acquisition method
correction in the expression level of a target miRNA in the second and subsequent sample is performed using a correction coefficient for the second and subsequent sample, respectively. That is, when the second analyte correcting the expression level of the target miRNA, the correction coefficient (c2 for the second analyte 1-1 or c2 1-1 using '), a target for the third sample in correcting the miRNA expression level correction coefficient (c3 for the third analyte 1-1 or c3 1-1 using a ').
[0108] As a correction factor, when using a difference obtained by subtracting the representative value of the correction endogenous miRNA second subsequent sample from the representative value of the correction endogenous miRNA reference analyte, i.e., if the above equation 1, the second and subsequent by adding the correction factor to the logarithm of the measured values ​​of each target miRNA expression level in the specimen, the correction of the expression level of a target miRNA for each specimen of the second and subsequent steps are performed. Expressing correction in this case by the formula, the corrected expression level Ei of the i-th miRNA in some "analyte to be corrected" is calculated according to the following formula 9.
[0109] [Number 9]

[0110] Here, Wi is the signal measurements from the probe immobilization regions to capture the i th miRNA.
[0111] On the contrary, as the correction coefficient, from the representative value of the correction endogenous miRNA second and subsequent sample, when using a difference obtained by subtracting the representative value of the correction endogenous miRNA reference analyte, i.e. the formula 1 ' in the case of, by subtracting the correction factor from the logarithm of the measured values ​​of each target miRNA expression level in a specimen of the second and subsequent correction of the expression level of the target miRNA are performed for each sample in the second and subsequent . Expressing correction in this case the formula is corrected expression level Ei of the i-th target miRNA in the "analyte to be corrected" can be determined by the following equation 9 '.
[0112] [Formula 10]

[0113] Here, the definition of Wi are the same as above formula 9.
[0114] If the corrected target miRNA expression levels measured in the second sample, or added to c2 to the logarithm of the measured values ​​of each target miRNA expression in the second sample, or if subtracting the c2 ' good. The same applies to the sample of the third and subsequent. Although the difference between the representative value and the reference value of the first analyte and the reference analyte is naturally 0, the structure of the program, the computation of adding or subtracting the zero target miRNA expression level of the first sample no problem be carried out.
[0115] 1-2. Fixed value correcting method
correction target miRNA 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 miRNA expression levels for a specimen, the correction coefficient (r for the sample 1-2 or r 1-2 using a ').
[0116] As a correction factor, when using a difference obtained by subtracting the representative value of the correction endogenous miRNA analyte from the reference value, i.e., if the above expression 3, the correction to the logarithm of the measured value of the expression level of each target miRNA in the sample by adding the coefficient correction of the expression level of a target miRNA for each analyte it is performed. Expressing correction in this case by the formula, the corrected expression level Ei of the i-th target miRNA in some "analyte to be corrected" can be determined by the following equation 10.
[0117] [Number 11]

[0118] Here, Wi is the signal measurements from the probe immobilization regions to capture the i th miRNA.
[0119] On the contrary, as the correction coefficient, when using a difference obtained by subtracting the fixed number from the representative value of the correction endogenous miRNA analyte, that is, when the above equation 3 ', the measurement of the target miRNA expression in specimens by subtracting the correction factor from the pairs of values ​​numerical correction of the target miRNA expression levels for each sample is performed. Expressing correction in this case the formula is corrected expression level Ei of the i-th target miRNA in the "analyte to be corrected" is calculated according to the following formula 10 '.
[0120] [Number 12]

[0121] Definition of Wi is the same as in the above formula 10.
[0122]
correction step-2 is a method of correcting the expression level of the target miRNA by using a correction coefficient obtained by the correction coefficient acquiring step-2, divided by the correction factor the expression level of a target miRNA 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.
[0123] 2-1. Reference analyte acquisition method
correction in the expression level of a target miRNA 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 miRNA for the second analyte correction coefficient (c2 for the second analyte 2-1 or c2 2-1 using '), a target for the third sample in correcting the miRNA expression level correction coefficient (c3 for the third analyte 2-1 or c3 2-1 using a ').
[0124] As a correction coefficient, a representative value of the correction endogenous miRNA second subsequent sample to be corrected as the denominator, the case of using the ratio of the representative value and the molecules of the correction endogenous miRNA reference analyte, i.e. of formula 5 in this case, by multiplying the correction coefficient to the logarithm of the measured values ​​of each target miRNA expression level in a specimen of the second and subsequent correction of the expression level of a target miRNA for each specimen of the second and subsequent steps are performed. Expressing correction in this case by the formula, the corrected expression level Ei of the i-th target miRNA in some "analyte to be corrected" is calculated according to the following formula 11.
[0125] [Formula 13]

[0126] Here, Wi is the signal measurements from the probe immobilization regions to capture the i th miRNA.
[0127] On the contrary, as the correction coefficient, when the representative value of the correction endogenous miRNA reference analyte as the denominator, is used the ratio of the representative value and the molecules of the correction endogenous miRNA second and subsequent sample, that is, the in the case of formula 5 'is by dividing the logarithm of the measured values ​​of each target miRNA expression level in a specimen of the second and subsequent correction factor, the correction of the expression level of a target miRNA for each specimen of the second and subsequent It is carried out. Expressing correction in this case by the formula, the corrected expression level Ei of the i-th miRNA in some "analyte to be corrected" is calculated according to the following formula 11 '.
[0128] [Number 14]

[0129] Here, the definition of Wi are the same as the formula 11.
[0130] If the corrected target miRNA expression levels measured in the second sample, c2 to the logarithm of the measured values of each target miRNA expression in the second sample 2-1 or 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 miRNA is the same. Although the ratio of the fixed numerical (reference value) in the representative value and the method of the first analyte and the reference analyte is naturally 1, the structure of the program, one to the target miRNA expression level of the first sample multiplying or target miRNA expression level no problem be carried out calculations that divide by 1.
[0131] 2-2. Fixed value correcting method
correction target miRNA 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 miRNA expression levels for a specimen, the correction coefficient (r for the sample 2-2 or r 2-2 using a ').
[0132] As a correction coefficient, a representative value of the correction endogenous miRNA analyte as the denominator, the case of using the ratio of the reference value was set to molecules, that is, when the equation 7, the measured value of the expression level of each target miRNA in the sample by multiplying the correction coefficient to the logarithm, the correction of the expression level of a target miRNA for each analyte are performed. Expressing correction in this case by the formula, the corrected expression level Ei of the i-th target miRNA in some "analyte to be corrected" can be determined by the following equation 12.
[0133] [Number 15]

[0134] Here, Wi is the signal measurements from the probe immobilization regions to capture the i th miRNA.
[0135] On the contrary, as the correction coefficient, when the representative value of the correction endogenous miRNA analyte as the denominator, using the ratio of the reference value and molecules, that is, when the equation 7 ', each target miRNA in the sample by dividing the logarithm of the measured value of expression amount by the correction factor, the correction of the target miRNA expression levels for each sample is performed. Expressing correction in this case the formula is corrected expression level Ei of the i-th target miRNA in the "analyte to be corrected" is calculated according to the following formula 12 '.
[0136] [Number 16]

[0137] Here, the definition of Wi are the same as above formula 12.
[0138]
The corrected target miRNA expression levels, contrasting target miRNA expression levels between a plurality of bodily fluid analyte. Contrast between the case, since the target miRNA expression level of the first analyte and the reference analyte has not undergone correction, for example, a first analyte and a second analyte correction by the reference sample acquisition method is carried out It includes a target miRNA expression level of the first sample that has not been corrected, but the contrast between the target miRNA expression level of the corrected second sample, at least one of the analytes to be compared always It is corrected analyte. Therefore, "the corrected target miRNA expression levels, contrasting target miRNA expression levels between a plurality of bodily fluid analyte" The term, contrast between the corrected and have no reference specimen corrected other analytes embodiments are also included.
[0139] Comparative analysis process itself can be performed as in the conventional method. For example, it is possible to represent the comparative analysis results as a scatter diagram 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.
[0140] Further, the corrected target miRNA expression levels, one of the sample and the difference in the expression level of a target miRNA in between the rest of the other analytes calculated, logarithmized fold change with the difference (fold-change it may represent a comparative analysis result). For example, the expression level of a target miRNA in the reference sample (for reference analyte acquisition method) or the expression level of the corrected target miRNA in the first sample and (in the case of fixed-value correcting method), has been corrected in the specimen of the second and subsequent the difference between the expression level of the target miRNA to 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.
[0141] MiRNA expression analysis apparatus of the present invention is an apparatus for carrying out the comparative analysis method of the present invention,
measured for a plurality of body fluid analytes, correction shown in measurements and SEQ ID NO: 1-10 in the expression level of the target miRNA storage means for storing the measured value of expression amount of one or more correction endogenous miRNA chosen from use endogenous miRNA;
for each fluid sample, from the measured values of one or more of the expression amount of the correction endogenous miRNA , the representative value, preferably to obtain a representative value expressed in logarithm, the representative value acquiring means;
and a reference value which is arbitrarily set with respect to the expression level of the correction endogenous miRNA, acquired by the representative value acquiring means the Samata the ratio of the representative value of each body fluid sample, to obtain respectively a correction coefficient of the target miRNA expression level for each body fluid sample, the correction coefficient acquiring section;
using each correction coefficient acquired by the correction coefficient acquiring section Te, each such body fluids Correcting the expression level of the measured target miRNA in the body, correcting means; and
the corrected target miRNA expression levels, an output means for outputting the result of comparing the target miRNA expression level between at least two fluid sample
containing .
[0142] In one certain embodiment, the reference value is a representative value of the correction endogenous miRNA of the first specimen is arbitrarily selected (reference samples), the expression level of a target miRNA measured in a body fluid sample of the second and subsequent There is corrected. That is, in this embodiment, miRNA expression analysis apparatus of the present invention,
for each of a plurality of analytes, the expression level of a plurality of target miRNA measured simultaneously and one or more of the measured value of expression amount of the correction endogenous miRNA a storage storing unit;
for each fluid sample, from the measured value of expression amount of the correction endogenous miRNA, representative value, preferably to obtain a representative value expressed in logarithm, the representative value acquiring means and;
arbitrarily selected the first sample a reference analyte that is, a representative value of the correction endogenous miRNA reference analyte as a reference value, the difference between the correction endogenous miRNA representative value of the reference value and the remaining second and subsequent analyte or the ratio, respectively acquires a correction coefficient for the second and subsequent sample, and the correction coefficient acquiring section;
using a correction coefficient for the second and subsequent sample obtained by the correction coefficient acquiring section respectively, the second measurement in subsequent specimen Correcting the expression level of a target miRNA that is, the correction means and;
the corrected target miRNA expression levels, an output means for outputting the result of comparing the target miRNA expression level between at least two fluid sample
containing.
[0143] In another embodiment, the reference value is a fixed value that is arbitrarily defined in terms correction endogenous miRNA expression levels, for all the samples including the first sample, is carried out to correct the target miRNA expression levels. That is, in this embodiment, miRNA expression analysis apparatus of the present invention,
for each of a plurality of body fluid analytes, simultaneously measured expression level of a plurality of target miRNA and one or more correction endogenous expression of measured values of miRNA ; storage means and for storing
for each specimen, the measured value of expression amount of the correction endogenous miRNA, representative value, preferably to obtain a representative value expressed in logarithm, the representative value acquiring means and;
fixed numerical ; as a reference value, the Samata the ratio of representative values of the correction endogenous miRNA of the reference values and the sample, corrected to obtain respectively as a coefficient, the correction coefficient acquiring means and for the sample
obtained by the correction coefficient acquiring means respectively using a correction factor for each analyte which is to correct the expression level of a target miRNA measured in each sample, and correction means;
corrected by the target miRNA expression level, of at least two bodily fluid analyte In and output means for outputting a result of comparing the target miRNA expression levels
including.
[0144] A block diagram showing an outline of an example of a configuration of the analysis apparatus of the present invention shown in FIG. Analysis device 10 of the present invention, the 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 expression level of a target miRNA in accordance with the present invention.
[0145] 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. Expression level data obtained by hybridization assays using microarrays, for example, the apparatus of the present invention is read by the reading means, such as another scanner is converted into numerical data, the numerical data from the input unit 110 is 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.
[0146] Expression level data digitized read input from the input unit 110 expression amount data, or by built-in reading means to the analyzer 10 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 measured expression level of a plurality of target miRNA was and one or more expression level of the correction endogenous miRNA simultaneously.
[0147] Measurement data of the expression amount of the target miRNA and the correction endogenous miRNA of each sample stored in the storage unit 140, the conversion unit 160, the bottom is converted to 2 logarithm. Then, the analysis unit 170, for each sample, representative values ​​of log-transformed expression levels of the measured value of the correction endogenous miRNA is obtained. Representative value, even when using as described in the description of the comparative analysis, for example, one or more correction endogenous miRNA expression level of the average value or median value only (one type of correction endogenous miRNA to correction, when the probe immobilization regions to measure it are a plurality present on the array, the representative value can be a mean or median), or a measurement of the particular one of the correction endogenous miRNA obtain.
[0148] After the representative values ​​are acquired, the analyzing unit 170, Samata between the representative value and the representative value of the correction endogenous miRNA second and subsequent analytes correction endogenous miRNA reference analyte ratio is calculated, the first correction factor for the 2 subsequent sample is obtained, respectively. Details of obtaining the correction factor is as described in comparison analysis. Incidentally, 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 coefficient 1 no problem be configured so as to acquire (if calculating the ratio).
[0149] In the apparatus 10, selection of the reference specimen can be carried out by a person operating the apparatus 10 to specify any one sample from the input unit 110. Alternatively, it may be elected 1 specimen apparatus 10 is automatically reference analyte. 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.
[0150] Then, the analysis unit 170, using the correction coefficient for the second and subsequent sample, respectively, to correct the expression level data of the target miRNA measured in the sample of the second and subsequent. Correction operation details are as described in comparison analysis. Incidentally, the structure of the program, for the target miRNA expression level data of the first analyte which is a reference analyte, correction using the correction coefficient 0 (if calculates a difference) or the correction factor 1 (the case of calculating the ratio) no problem is also possible to perform the operation.
[0151] Then, the analysis unit 170, a target miRNA expression level of the reference sample, the contrast between the corrected target miRNA expression level of the second and subsequent sample is performed. The results of the comparison is the output unit 130, is output to the display unit 120, is displayed. Further, the output device or a recording medium such as the comparison result of the printers can be output. Furthermore, the output unit 130 may be configured to output a comparison analysis result to an external storage device such as a database, that exists outside of the apparatus via a network.
[0152] Storage unit 140, in addition to storing the measured value of expression amount of expression levels and a plurality of correction endogenous miRNA of multiple target miRNA, intermediate analysis results stored appropriately produced in each step of the above.
[0153] 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.
[0154] Further, in the analyzer, instead of using the correction endogenous miRNA representative value of the reference sample as a reference value, the device 10, it may be registered fixed number specified in advance in the conversion unit 160 or the like, in the reference sample it may be used as a reference value in place of the representative value of the expression amount of the correction endogenous miRNA. In this case, details of the method, comparison analysis, are as described .
[0155] Further, the present invention provides a program for causing a computer to function as the analysis apparatus described above. The program, specifically, the means described above the computer (i.e., the storage unit, the representative value acquiring unit, the correction coefficient acquiring means, correcting means, and output means) is a program for functioning as a. Alternatively, the present invention provides a program for executing the steps of the comparative analysis method of the present invention described above to a computer. The comparative analysis method, the measurement step described above, the representative value acquiring step, the correction coefficient acquiring step, and the correction step includes further the corrected target miRNA expression levels, the target miRNA expression levels between a plurality of bodily fluid analyte comparative analysis step of contrasting may include. These programs, using the data of the expression amount of the correction for endogenous miRNA measured simultaneously with the expression level of the target miRNA by microarray or the like, a program for executing the correction of the expression level of a target miRNA in the computer.
[0156] Furthermore, the present invention is recorded with the above-mentioned one of the programs, and a computer-readable recording medium.
[0157] "Recording medium", a flexible disk, a magneto-optical disk, ROM, EPROM, EEPROM, CD-ROM, MO, may 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."
[0158] 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.
[0159] The present invention is a probe for capturing a probe and a plurality of correction endogenous miRNA for capturing a plurality of target miRNA comprises immobilized support provides chips for miRNA expression analysis. Preferred conditions for the chip is as described in the comparative analysis method of the present invention.
[0160] In the present invention, at least one of miRNA of SEQ ID NO: 1 to 10 described below, for use as correction endogenous miRNA.
[0161] SEQ ID NO: 1 is the nucleotide sequence of hsa-miR-6085, registered in the Accession No. MIMAT0023710 the miRBase. As used herein, the term "miR-6085 gene" or "miR-6085" encompasses such hsa-miR-6085 and other species homolog or ortholog of SEQ ID NO: 1. hsa-miR-6085 gene can be obtained Voellenkle C et al., 2012, RNA, 18 vol., by the method described in P.472-484.
[0162] SEQ ID NO: 2 is the nucleotide sequence of hsa-miR-1227-5p that is registered with Accession No. MIMAT0022941 the miRBase. As used herein, the term "miR-1227-5p gene" or "miR-1227-5p" encompasses such hsa-miR-1227-5p or other species homologs or orthologs of SEQ ID NO: 2. hsa-miR-1227-5p gene can be obtained Berezikov E et al, 2007, Molecular Cell, 28, pp. by the method described in P.328-336.
[0163] SEQ ID NO: 3 is the nucleotide sequence of hsa-miR-2861, registered in the Accession No. MIMAT0013802 the miRBase. As used herein, the term "miR-2861 gene" or "miR-2861" encompasses such hsa-miR-2861 and other species homolog or ortholog of SEQ ID NO: 3. hsa-miR-2861 gene, Li H, et al., 2009, Journal of Clinical Investigation, 119 vol may be obtained by the method described in P.3666-3677.
[0164] SEQ ID NO: 4 is the nucleotide sequence of hsa-miR-149-3p, registered in Accession No. MIMAT0004609 the miRBase. As used herein, the term "miR-149-3p gene" or "miR-149-3p" encompasses such hsa-miR-149-3p and other species homolog or ortholog of SEQ ID NO: 4. hsa-miR-149-3p gene, Lagos-Quintana M et al., 2002, Current Biology, 12 vol., can be obtained by the method described in P.735-739.
[0165] SEQ ID NO: 5 is the nucleotide sequence of hsa-miR-4463, registered in the Accession No. MIMAT0018987 the miRBase. As used herein, the term "miR-4463 gene" or "miR-4463" encompasses such hsa-miR-4463 and other species homolog or ortholog of SEQ ID NO: 5. hsa-miR-4463 gene, Jima DD et al., 2010, Blood, 116 vol, may be obtained by the method described in p.e118-e127.
[0166] SEQ ID NO: 6 is the nucleotide sequence of hsa-miR-4508, registered in the Accession No. MIMAT0019045 the miRBase. As used herein, the term "miR-4508 gene" or "miR-4508" encompasses such hsa-miR-4508 and other species homolog or ortholog of SEQ ID NO: 6. hsa-miR-4508 gene, Jima DD et al., 2010, Blood, 116 vol, may be obtained by the method described in p.e118-e127.
[0167] SEQ ID NO: 7 is the nucleotide sequence of hsa-miR-6090, registered in the Accession No. MIMAT0023715 the miRBase. As used herein, the term "miR-6090 gene" or "miR-6090" encompasses such hsa-miR-6090 and other species homolog or ortholog of SEQ ID NO: 7. hsa-miR-6090 gene can be obtained Yoo JK et al., 2012, Stem Cells and Development, 21 vol., by the method described in P.2049-2057.
[0168] SEQ ID NO: 8 is the nucleotide sequence of hsa-miR-6775-5p that is registered with Accession No. MIMAT0027450 the miRBase. As used herein, the term "miR-6775-5p gene" or "miR-6775-5p" encompasses such hsa-miR-6775-5p or other species homologs or orthologs of SEQ ID NO: 8. hsa-miR-6775-5p gene can be obtained Ladewig E et al, 2012, Genome Research, 22 vol., by the method described in P.1634-1645.
[0169] SEQ ID NO: 9 is the nucleotide sequence of hsa-miR-6803-5p that is registered with Accession No. MIMAT0027506 the miRBase. As used herein, the term "miR-6803-5p gene" or "miR-6803-5p" encompasses such hsa-miR-6803-5p or other species homologs or orthologs of SEQ ID NO: 9. hsa-miR-6803-5p gene can be obtained Ladewig E et al, 2012, Genome Research, 22 vol., by the method described in P.1634-1645.
[0170] SEQ ID NO: 10 is the nucleotide sequence of hsa-miR-5787, registered in the Accession No. MIMAT0023252 the miRBase. As used herein, the term "miR-5787 gene" or "miR-5787" encompasses such hsa-miR-5787 and other species homolog or ortholog of SEQ ID NO: 10. hsa-miR-5787 gene, Yoo H, et al., 2011, Biochem Biophys Res Commun, 415 vol, may be obtained by the method described in P.567-572.
Example [0171] Hereinafter, more specifically explained on the basis of the present invention embodiment. However, the present invention is not limited to the following examples.
[0172]
by the following process was selected correction endogenous miRNA for use in the present invention.
[0173] (DNA Microarray)
using Toray Co., Ltd. "3D-Gene" human miRNA oligo chip (miRBase release 20 compatible) the following experiment was conducted.
[0174] (Sample RNA Preparation)
as a sample RNA, from a healthy human serum 157 specimens, using RNA extracted using "3D-Gene" RNA extraction reagent from liquid sample kit. The resulting sample RNA, were labeled with "3D-Gene" miRNA labeling kit ( Toray). Labeled specimen RNA, 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 AUTO setting.
[0175] (MiRNA acquisition signal value)
of the signal value of the miRNA obtained from DNA microarray, the bottom is converted to 2 logarithm, experiments were conducted error correction by external standard nucleic acid added during RNA extraction and labeling.
[0176] (Correction acquisition of endogenous miRNA)
The obtained experimental error corrected miRNA signal values of all the 157 specimens was extracted analyte between minimal variation miRNA by geNorm method. GeNorm method is a search method of the endogenous housekeeping gene (reference gene) in quantitative RT-PCR method proposed by Vandesompele et al (Genome Biology 2002, 3: research0034 -research0034.11). Used as a method of selecting a mRNA which is specific endogenous housekeeping gene in the sample of interest. Specifically in the same specimen, the signal values of all the detection target gene, to calculate the ratio of the signal value of the two genes in brute. Next, all combinations obtained in the ratio of the values of the gene for the (A) to calculate the standard error between the analyte. Next, for a particular gene, to calculate the sum of the standard error obtained in relation to other genes (V) and (M). Gene The M value is small, are excellent endogenous housekeeping gene.
[0177] On the other hand, A and V are obtained by the above calculation method, as the signal value is small, becomes a small value, are the apparent good endogenous housekeeping gene. DNA microarrays used here, in order to comprehensively detect all miRNA registered in miRBase release 20, when implementing the geNorm method using this data, the miRNA is small signal value would preferentially selected. From this phenomenon, it has been avoided by selecting the endogenous housekeeping gene in the DNA microarray detected by hitherto geNorm method.
[0178] In this embodiment, among the miRNA detected by DNA microarray, the signal value is greater than 64 in more than half of all of the samples were selected only miRNA to be detected stably as an object of application of the geNorm method. Furthermore the characteristics of geNorm method, since it is selected endogenous housekeeping genes based on the gene expression level ratios in the same sample, the gene selection showing a stable expression amount between samples is significantly expression pattern different Given the difficulties, the pre experimental error corrected each miRNA signal values ​​of all the 157 specimens to obtain CV (standard error / mean), CV selects only miRNA is 0.1 or less as a target of geNorm method. As a result, of the signal values ​​obtained from the 2555 one miRNA detection probe was considered appropriate to the signal value of the probe for detecting the 281 one miRNA target of geNorm method.
[0179] Indeed from 281 one miRNA applying the geNorm method, small endogenous housekeeping gene M value, i.e. were selected 10 kinds can be used as a correction for endogenous miRNA. SEQ ID NO: of the 10 kinds of correction endogenous miRNA, Table miRNA name of, MIMAT registered in miRBase number, calculated M value geNorm, nucleotide sequence, and the proportion of guanine cytosine in sequence (GC%) It is shown in 1.
[0180] [Table 1]

[0181] Detection of miRNA by DNA microarrays, because they are made by hybridizing method for forming a probe with a nucleic acid duplex to capture miRNA and its target miRNA targets, containing guanine and cytosine for stabilizing the nucleic acid duplex the amount is higher sequence is particularly stable manner expected to be detected by DNA microarray. In other words, guanine and high content sequence of cytosine is generally assumed when preferentially selected as an endogenous housekeeping gene. However, as shown in Table 1, necessarily endogenous housekeeping genes selected by geNorm method (correction endogenous miRNA), the content of guanine and cytosine is not so high. The content of guanine and cytosine is higher miRNA was not observed phenomenon becomes preferentially endogenous housekeeping gene (corrected for endogenous miRNA) reversed.
[0182]
Using the selected correction for endogenous miRNA singly was expression amount correction of a target RNA of the plurality of body fluid analytes.
[0183] From physical instability of RNA, when measuring the RNA expression levels, the quality is different, i.e., between analyte degradation of the RNA are different, it is often the case that the required absolute expression level comparisons. Especially if the specimen is a body fluid, it is the case of obtaining something like of results by measuring the target miRNA expression of the long-time in the sample placed in room temperature is assumed.
[0184] At this time, by performing a specific correction corresponding to RNA degradation degree the amount of correction for endogenous miRNA as criteria contained in the serum, irrespective of the RNA quality, miRNA expression profiles of (expression relative miRNA the amount ratio), is considered to be compared between absolute analyte becomes possible.
[0185] In this embodiment, by storing the serum from a human with various conditions, the quality of the miRNA contained in the serum to create a serum at various levels, the measured value of the extracted RNA specimens from these sera and applying the correction method of the present invention for.
[0186] (DNA Microarray)
Toray Co., Ltd. "3D-Gene" human miRNA oligo chip (miRBase release 20 corresponding, Toray Inc.) was subjected to the following experiments using.
[0187] (Sample RNA Preparation)
bled human serum, and allowed to stand after the serum separated into the following two types of conditions, changing the quality of the RNA contained in the serum. The two conditions are shown below. 1: allowed to stand in room temperature for 6 hours after serum separation, perform RNA extraction. 2: standing for 24 hours 25 ° C. After serum separation, perform RNA extraction. Total RNA was extracted using "3D-Gene" RNA extraction reagent from liquid sample kit RNA from the serum that was created in each condition were used as specimens RNA.
[0188] Each resulting sample RNA was labeled using "3D-Gene" miRNA labeling kit (Toray). Labeled specimen RNA, in accordance with standard protocols of "3D-Gene" human miRNA oligo 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 AUTO setting.
[0189] (Correction of miRNA signal value)
of the signal value of the miRNA obtained from DNA microarray, showing the distribution of signal values bottom is converted to 2 logarithm (i.e. expression level signal value of uncorrected) in Figure 4A. In contrast, the signal value of the correction endogenous miRNA shown in SEQ ID NO: 1-10 show corrected by the reference value is a fixed value that has been set in advance separately, the distribution of the corrected signal values in Figure 4B ~ K . In the figure, Day-0 (broken line) is the analyte RNA condition 1, Day-1_25 ℃ (solid line) is the analyte RNA condition 2.
[0190] Here, the correction according to the signal value of the correction endogenous miRNA shown in SEQ ID NO: 1-10 were carried out by the method shown below.
[0191] Correction for endogenous miRNA shown in SEQ ID NO: 1-10, and set a fixed value that is used as the respective reference values. Further, for each sample, the bottom signal values ​​of the correction endogenous miRNA shown in SEQ ID NO: 1-10 is converted to 2 logarithm, taking the ratio of the reference value signal value after the conversion as the representative value (reference value / representative value), and the correction coefficient. Correction factor will have been set for each correction endogenous miRNA of SEQ ID NO: 1-10. These correction factors, each correction endogenous miRNA, was corrected by multiplying all of the miRNA signal values ​​for each analyte.
[0192] As a result, as shown in Figure 4B ~ K, using any of the correction endogenous miRNA shown in SEQ ID NO: 1-10 alone, it is identical the expression profile of two samples with different conditions It was possible.
[0193]
As shown in Example 2, the correction endogenous miRNA of SEQ ID NO: 1-10, but each with the effect of correcting the expression level of either alone sufficient targeted miRNA, by further combining these, in some cases the loss of part of the correction endogenous miRNA data by deficiencies such experiments happened, it is envisioned that can complement this. Therefore, performs correction of the expression level of the target miRNA by using a combination of a plurality of correction endogenous miRNA, it confirmed the effect of the correction. Specimen used was the same as in Example 2.
[0194] The signal value of the correction endogenous miRNA of SEQ ID NO: 2, 4, and 5 bottom is converted to 2 logarithm, with the mean or median signal value after the conversion, and the representative value. The reference value using a fixed value. The average value of all of the expression amount of the three kinds of correction endogenous miRNA in a plurality of serum RNA sample comprising two specimens prepared in Example 2 (after logarithmic conversion signal value), the fixed numerical value and the average value It was used as the (reference value). For each sample, taking the ratio of the representative value and the reference value of the correction endogenous miRNA (reference value / representative value) and the correction coefficient for the analyte. The correction factor, by multiplying all of the target miRNA signal values ​​each corresponding specimen was corrected target miRNA signal values.
[0195] 5, in the case of using an average value of the after-log signal value as a representative value of the expression amount of the correction endogenous miRNA, is the distribution of the corrected signal value of the target miRNA. As in Example 2 was corrected by using the correction endogenous miRNA of SEQ ID NO: 1-10 respectively alone, even when subjected to correction using a combination of a plurality of correction endogenous miRNA, for each sample target the miRNA expression profiles it was possible to identification with.
[0196]
As shown in Example 3, the correction endogenous miRNA of SEQ ID NO: 1-10 are useful for the correction by combining these. This compensation effect was confirmed to show a similar effect by combining any of SEQ ID NO: 1-10. Specimen used was the same as in Example 2.
[0197] Bottom signal values ​​of the correction endogenous miRNA of SEQ ID NO: 2, 3, 4, 5 and 6 are converted to two's log, for each sample, the signal value after the correction endogenous miRNA conversion of any of the three obtaining an average value, and a representative value in the sample. Reference value is a fixed numerical value, the after-log signal values ​​of the three correction endogenous miRNA using the representative value acquiring an average value of a plurality of serum RNA sample comprising two specimens prepared in Example 2 It was used as a fixed numeric value (reference value). For each sample, taking the ratio of the representative value and the reference value (reference value / representative value) and the correction coefficient for the analyte. The correction factor is multiplied by all of the target miRNA signal values ​​each corresponding sample were corrected miRNA expression level of each sample.
[0198] The distribution of the corrected signal values ​​shown in FIG. 8A is a distribution of the expression level signal value of uncorrected Figure 8B ~ I is a distribution of the corrected expression level signal values. As in Example 2 was corrected by using the correction endogenous miRNA of SEQ ID NO: 1-10 respectively alone, the expression profile of a target miRNA in the sample was able to identification with.
[0199]
when examined in Example 1, in the step of labeling with each of the sample RNA obtained from serum RNA, "3D-Gene" miRNA labeling kit ( Toray), externally added two synthetic RNA sequence as a reference substance (each 20 mer, referred to as spike control 1 and spike control 2) was added. These synthetic RNA sequences are fluorescently labeled as with serum-derived miRNA, signal intensity by "3D-Gene" human miRNA oligo chip ( Toray Industries, Inc.) was detected. Therefore, each 8-point spike control 1 and spike control 2, a signal value obtained from the spot of a total of 16 points the bottom is converted to 2 logarithm, to obtain the median was the representative value. And the representative value, taking the ratio of the fixed numerical value preset (reference value) (representative value of the reference value / each analyte) to obtain a correction coefficient for each sample. The correction factor, correction is performed by multiplying all of the miRNA signal values for each analyte. The distribution of the signal value before correction in Fig. 6A, shows a distribution of signal values after the correction in FIG. 6B. In accordance with correction added spike controls from outside during labeling, the signal value of the target miRNA in the sample was shown to be not sufficiently corrected.
[0200]
To date is shown as a expression level correction prior examples miRNA, let-7d-5p, let-7g-5p, let-7i-5p, miR-16, miR-31, miR- of 223, the amount of expression in the second embodiment shown in Table 2. except has-miR-16-5p, when the bottom of the resulting signal value is converted into 2 logarithm, only shown signals in the range of 0-5, on the grounds that the expression amount is not sufficient, the expression of all miRNA the criteria for correcting the amount was not suitable. Moreover, the distribution of the correction signal value when corrected by hsa-miR-16-5p in FIG. The correction by the expression level of hsa-miR-16-5p, the signal value of the target miRNA in the sample was shown to be not sufficiently corrected.
[0201] [Table 2]

DESCRIPTION OF SYMBOLS
[0202] 10 device
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 comparative analysis of the expression level of a target miRNA across multiple bodily fluid analyte,
the plurality of body fluid analytes, are selected from the correction endogenous miRNA shown in measurement and SEQ ID NO: 1-10 in the expression level of the target miRNA performing one or more of the measurement of the expression amount of the correction endogenous miRNA simultaneously measuring step;
for each body fluid sample, one or more correction endogenous chosen from the correction endogenous miRNA shown in SEQ ID NO: 1-10 obtaining a representative value from the measured values of miRNA expression levels, the representative value acquiring step;
representatives of the arbitrarily set reference values for the expression amount of the correction endogenous miRNA, each body fluid sample obtained by the representative value acquiring step the Samata ratio of the value, the target acquires respectively as a correction coefficient for miRNA expression levels, the correction coefficient acquiring step for each fluid sample; and
using respectively the acquired correction coefficient for each body fluid sample, the fluid measured in the sample Correcting the expression level of the target miRNA was, correction step
including the method.
[Claim 2] 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 miRNA,
in (b) the correction coefficient acquisition step, when obtaining 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 miRNA,
(c) the correction in 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 miRNA, or
(d) in the correction coefficient acquiring 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 miRNA,
by performing the correction method according to claim 1.
[Claim 3] The reference value is fixed numbers are arbitrarily defined with respect to the correction endogenous miRNA expression levels, or, correction endogenous miRNA expression levels obtained for the first bodily fluid sample arbitrarily selected from the plurality of bodily fluid analyte a representative value, method according to claim 1 or 2.
[Claim 4] Said determining step, one or more correction endogenous miRNA is selected from the correction endogenous miRNA shown in the nucleic acid probe and SEQ ID NO: 1-10 for capturing a plurality of target miRNA immobilized on a support probe, a nucleic acid sample derived from the labeled body fluid sample with a labeling substance is contacted performs hybridization, the target miRNA and the one or each signal intensity expression levels of a plurality of correction endogenous miRNA to capture and obtaining a measurement value, the method according to any one of claims 1 to 3.
[Claim 5] It said body fluid sample is blood, serum or plasma, the method according to any one of claims 1 to 4.
[Claim 6] The representative value is calculated from the measured values ​​of one or more correction endogenous miRNA expression levels are selected from the correction endogenous miRNA shown in SEQ ID NO: 1-10, the mean or median was represented by logarithm the value a method according to any one of claims 1 to 5.
[Claim 7] A miRNA expression analysis device for comparative analysis of the expression level of a target miRNA in a plurality of body fluid analytes,
measured for a plurality of bodily fluid analyte, the correction endogenous shown in measurements and SEQ ID NO: 1-10 in the expression level of the target miRNA storage means for storing the measured value of expression amount of one or more correction endogenous miRNA is selected from sexual miRNA;
for each body fluid sample, 1 or is selected from the correction endogenous miRNA shown in SEQ ID NO: 1-10 obtaining a representative value from the measured value of expression amount of a plurality of correction endogenous miRNA, the representative value acquiring unit;
acquired by the reference value which is arbitrarily set with respect to the expression level of the correction endogenous miRNA, wherein the representative value acquiring means the Samata the ratio of the representative value of each body fluid sample that is the target to obtain respectively a correction coefficient for miRNA expression levels, the correction coefficient acquiring means for each fluid sample;
a correction coefficient obtained for each body fluid sample using each , To correct the expression level of the measured target miRNA in the body fluid sample, the correction means; and
the corrected target miRNA expression levels, and outputs the result of comparing the target miRNA expression level between at least two fluid sample output It means
including said device.
[8.] It said correction means,
(a) in 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 miRNA,
in (b) 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 miRNA,
(c) the correction in the coefficient obtaining means, when acquiring a 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 miRNA, or
(d) in the correction coefficient acquiring section , 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 miRNA,
it performs the correction by apparatus of claim 7.
[Claim 9] Wherein stored in the storage unit, one or more correction endogenous miRNA is selected from the correction endogenous miRNA shown in measurements and SEQ ID NO: 1-10 in the expression level of the target miRNA contained in each of a plurality of bodily fluid analyte measurement of the expression level, a probe for capturing the probe and said one or more correction endogenous miRNA for capturing a plurality of target miRNA immobilized on a support, labeled with a labeling substance contacting the nucleic acid sample from a body fluid specimen subjected to hybridization, a value measured respectively the expression level as the signal strength measurements of the target miRNA and the one or more correction endogenous miRNA, claim 7 or according to 8.
[Claim 10] To compare analyzing the expression level of a target miRNA across multiple bodily fluid analyte, one or more computers,
for each of a plurality of bodily fluid analyte, the correction endogenous shown in measurement and SEQ ID NO: 1-10 in the expression level of the target miRNA performing one or more of the measurement of the expression amount of the correction endogenous miRNA simultaneously selected from sexual miRNA, measuring step;
for each body fluid sample, 1 or is selected from the correction endogenous miRNA shown in SEQ ID NO: 1-10 obtaining a representative value from each measurement of the expression amount of the plurality of correction endogenous miRNA, representative value acquiring step;
and optionally set reference values for the expression amount of the correction endogenous miRNA, acquired by the representative value acquiring step the Samata the ratio of the representative value of each body fluid sample that is the target to obtain respectively a correction coefficient for miRNA expression levels, the correction coefficient acquiring step for each fluid sample; and
correction factors acquired for each body fluid sample using each , To correct the expression level of the measured target miRNA in the body fluid sample, the correction process
program for executing.
[Claim 11] To compare analyzing the expression level of a target miRNA across multiple bodily fluid analyte, one or a plurality of computers,
correction shown in measurements and SEQ ID NO: 1-10 in the expression level of the target miRNA contained in each of a plurality of bodily fluid analyte storage means for storing the measured value of expression amount of one or more correction endogenous miRNA chosen from use endogenous miRNA;
for each body fluid sample is selected from the correction endogenous miRNA shown in SEQ ID NO: 1-10 obtaining a representative value from the measured values of one or more of the expression amount of the correction endogenous miRNA, the representative value acquiring means;
and a reference value which is arbitrarily set with respect to the expression level of the correction endogenous miRNA, wherein the representative value acquiring means the Samata the ratio of the representative value of each body fluid sample obtained by, respectively acquires a correction coefficient of the target miRNA expression level for each body fluid sample, the correction coefficient acquiring means;
correction obtained for each body fluid sample using the coefficient, respectively Te, corrects the expression level of the measured target miRNA in the body fluid sample, the correction means; and
the corrected target miRNA expression levels, and outputs the result of comparing the target miRNA expression level between at least two fluid sample output means
program for functioning as a.
[Claim 12] To claim 10 or 11 records a program described, a computer-readable recording medium.
[Claim 13] The support probe and one or a plurality of probes for capturing the correction endogenous miRNA is selected from the correction endogenous miRNA shown in SEQ ID NO: 1-10 is immobilized to capture a plurality of target miRNA including, chip for miRNA expression analysis.