FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]
1. TITLE OF THE INVENTION: METHODS AND REAGENTS TO INACTIVATE NUCLEASES FOR PRESERVATION OF RIBONUCLEIC ACID
2. APPLICANT:
(a) Name: XCELRIS LABS LIMITED,
(A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956)
(b) Nationality: Indian
(c) Address: XCELRIS CORPORATE HEADQAUTERS,
OLD PREMCHAND NAGAR ROAD, OPP. STAYAGRAH CHHAAVANI BODAKDEV, AHMEDABAD-380054, INDIA

3.BACKGR0UND OF THE INVENTION FIELD OF THE INVENTION
The present invention relates largely to the fields of cell biology and molecular biology. More precisely, the invention discloses a novel method and compositions to preserve all types of ribonucleic acidespecially structural and functional ribonucleic acidin post harvested biological sample from endonucleases as well as exonucleases degradation.
DESCRIPTION OF RELATED ART
Field of cellular as well as molecular biology has enormous demand for obtaining high quality RNA. Intact messenger RNA, small RNA is essentiaf for identification of novel genes, transcribed variants in various biological cells and tissues during developmental phase, response to environment in prokaryotic and eukaryotic system. During given environmental conditions from normal versus diseases, biotic-abiotic stress leads to change in protein expression i.e in turn the RNA molecule turnover changes. Some RNA molecules degrade completely while other undergo partial degradation or supression wherein other undergo high expression. This difference capturing by using NGS technolgy, microarray or RTPCR leads to demarcation of various biological hypothesis. Therefore preserving the state of RNA molecule during and post sampling is very essential and basis of above mentioned studies. So herein we describe the composition which preserve the ribonucleic acidin post harvested biological sample to be used for deciphering important information about various mechanisms underlying intracellular events. Adequate measures are required for preservation of intactness of RNA molecules inside samples from sample collection to RNA purification. Cell lysis is predominantly prevalent during tissue collection that is a major factor responsible for RNA degradation via endogenous as well as exogenous nucleases . It is suggested that homeostasis of RNA molecule within cells is delicately balanced process that tends to be disrupted with the shift in metabolism occurs with tissue excision from its place. The process of tissue collection initiates one of the many processes where degradation of ribonucleic acid molecule takes place. In most of the cases, site of RNA isolation and harvesting of samples are distantly located for example in agri-biotechnology, plants are grown in field or greenhouse while they need to be brought to laboratory for further processing. If we need to capture the biological response like development al phases or specific yield in crop, in that case, tissue will be harvested at various stage and require storage untill experiment is completed,

therefore there is a need for preserving solution or media to protect RNA molecules in cells. We have observed that sample degradation leads to release of endogenous RNases which are responsible for degradation. Therefore there arises need for sample preservation to prevent the sample deterioration, followed by inactivation of the various types of RNases by means of coagulation, denaturation and inactivation of proteins by disrupting the protein structures in micro environment of cells. The alternate method of quick-freezing using liquid nitrogen is used to freeze the cell metabolism using liquid nitrogen followed by storage of samples at -80°C, this is a method to minimize the activity of RNases that are responsible for degradation of RNA. It is well known that intact high quality RNA can be obtained from frozen tissue samples, with minimum freezing thawing (US 7,517,697 B2). Transport and handling of liquid nitrogen is hazardous as well as required training, equipment and extra caution. Repeated freeze thawing process drastically decreases the quantity and quality of RNA molecule.
With the advent of next generation sequencing and molecular biology platforms, high quality genetic materials are required to understand the structural and functional role of nucleic acid molecules within harvested cells and tissues. The amount of nucleic acids especially ribonucleic acidrepresent the active population at given point of time, which provide insights into base level expression as well as elevated expression in experimental or disease conditions. Therefore it is important to protect the integrity of nucleic acids pertaining to their extractions.
Various prior art describes use of salts and buffers for RNA preservation . For destruction of RNases, Wolf et al., in 1970 described diethyl pyrocarbonate to be added to final concentration of 0.1% to molecular biology reagents, glassware or electrophoresis apparatus. The solutions are to be autoclaved for 20 mins to destroy RNase activity. Chomczynski, 1992 suggested storage of RNA in ethanol and use of guanidinium thiocyanate to inhibit RNase activity during RNA isolation(Sambrook et al., 1989). Favaloro et al in 1980, described the use of macleiod clay, and Berkenmeier et a! in 1979 described the use of Vanadyl-ribonucleioside complex to be used to inhibit Rnases during RNA Isolations from cells or tissues. Other reagents including SDS, EDTA, Protease have also been reported to have inhibitory effects during RNA isolation.

US Patent 7517697 describes water miscible solvents like ethyl acetate, acetic anyhydride, methoxy ethanol, ethanol to freeze biological samples. A large literature suggest ethanol, methanol, acetone, formaline in combination are best fixative for further recovery of DNA molecules from archived tissues. These solutions doesnot provide effectiveness in pursuing small ribonucleic acids at room temperature.
US Patent 2010/0028852 describes ammonium sulphate salt based compositions at 4°C to -80°C. The saturated solution described in composition precipitate at 4°C to -80°C and doesnot dissolve back. Therefore they have adverse effect on cell integrity and quantity and quality of RNAs. The composition aJso doesnt effectively preserve small RNAs.
Vincek et al., in US Patent 7138226 have described the use of 10% PEG and 90% methanol for preservation of tissue samples to carry out histological analysis using antigen by cognate antibdody and also effect on preserving the DNA and RNA in biological sample. Various literature reported the role of PEG in protein precipitation and its use also has been reported in stable protein formulation of purified isolated proteins. It has also been reported by Ingham et al 1981, that PEG donot have significant effect on protein melting temperature upto concentration of 30%wA/.
The potential for degradation lies in the amount of time required for collection of samples and processing the samples for homogenization with current methodologies, there is a high risk of RNA degradation. Also the process of freezing-thawing plays a major role in activation of RNases that degrade RNA. It is thought that ice damages the normal barriers formed between cellular compartments, so that nucleolytic enzymes in degradosomes, cytoplasmic vesicle, and extracellular regions are allowed ectopic access to cytoplasmic and nuclear RNA. US Patent No 5,256,571 reports a cell preservative solution comprising a water-miscibie alcohol in an amount sufficient to fix mammalian cells, water-miscible alcohol in an amount sufficient to fix mammalain cells, an anti-clumping agent and a buffering agent. Dimulescu et al., reports the apparent use of this fixative for preservation of RNA intergity in cancer cells.
The condition like salt concentration, pH and temperature, in a solution can have a dramatic affect on enzymatic catalysis. Despite the importance of salt effects on catalysis, their chemical origins are unknown for most enzymes. Various explanations have been proposed to explain the decrease in the catalytic activity. The present invention has a

composition, which also alter the intramolecular interactions between the binding sites of the enzyme, a conformational change of the enzyme or substrates, a decrease in "specfic1 interactions with substrates due to increased rigidity of the enzyme, and so forth. RNase A possess phenominal stability and can withstand extreme conditions like 0.25N sulfuric acid at 58°C, pH 3.0 at 95±100.8°C These harsh conditions can easily break noncovalent interactions but are unable to break the four disulfide bonds of RNase A, which are essential for secondary structure formation. Disulfide binds are covalent crosslinks in polypeptide chains. Crosslinks limit the number of unfolded conformations of a polypeptide chain, thereby destabilizing the unfolded state relative to the native state. If this loss of entropy in the unfolded state were the only disulfide bondmediated contribution to conformational stability, then disulfide bond-mediated stability would be reflected in the loop size, i.e. the number of amino-acid residues within the ring containing the disulfide bond. This model has also been applied to proteins with interweaving crosslinks, including RNaseA.
Disulfide bonds can be important for the function of a protein, as well as its conformational stability. Indeed, replacing a native disulfide bond with a pair of alanine residues can result in a variant protein that has greater conformational stability than does the wild-type protein. In other words, a native disulfide bond can actually destabilize the tertiary structure. Such disulfide bonds may be retained by natural selection to enable a particular function. RNase A catalyzes the cleavage of the P-O50 bond of RNA on the 30 side of pyrimidine residues to yield a 20,30-cyclic phosphodiester. His12 and His119 are the base and acid that mediate the transphosphorylation reaction. Lys41 assists in transition state stabilization. Replacing any of these three residues with alanine hinders catalysis by 104-to 105-fold. To achieve the maxima! rate of substrate cleavage, each activesite residue must be aligned precisely.
The composition of present invention described herein possess protective effects on tissue morphology, preserve intactness and integrity of ribonucleic acidmolecules, effective at low temperature for long term storage. Therefore innovation described herein is novel and nonobvious.
SUMMARY OF THE INVENTION
The given invention provides compositions and method for preserving total rionucleic acid in post harvested biological samples. The invention specifically deactivates proteins including ribonucleases that are responsible for degradation of ribonucleic acidmolecules.

ribonucleic acid in cells /tissue samples tend to degrade over a specific period of time if not processed properly. Use of present invention eliminates the need of liquid nitrogen at the site of collection or immediate freezing of samples. Also, it eliminates degradation of ribonucleic acid by Ribonucleic acidses due to constant thawing of freezed samples.
Samples covered in the present embodiment can be of prokaryotic micro-organisms as well as eukaryotic cells or tissues like plant roots, leaf, stem, inflorescence, flowers, fruits, immature seeds, mature seeds, animal tissues, human biopsy tissues. They are contemplated to be or to include cells, tissues, organs, and/or organisms. Samples may be obtained by any method such as cutting, piercing, chopping, etc, intact tissues (without damage are preferred) . The present invention also relates to proper storage or immersion of samples in compositions described in this invention for complete preservation of ribonucleic acid molecules. The samples can be of animal origin, for example whole organs brain, liver, heart, spleen, thymus, kidney, ovary, testis etc, or of plant origin like leaves, root, stem, infloroscence, apical bud etc. ribonucleic acid preservation of the tissue is enhanced if dissection of tissue is done within the solution of present invention.
In some embodiments, the ribonucleic acid preservation medium comprise of combination of sulphonic acid along with one or more polar or nonpolar organic solvents and in combination thereof. The preferred embodiment also consists polyamino carboxylic amino acid responsible for sequestring of divalent cations responsible for reduing catalytic activity of proteins.
In other embodiments of the solution, the ribonucleic acid stabilizing solution along with the sample is stored at -20°C to 25°C. More preferably, the sample is stored in the invention at low temprature, that enhances the preservation of ribonucleic acid. More precisely, ribonucleic acid can be stored in the present invention at low temprature for a long time.
Most of the biological processes are impaired by small changes in concentration of H+ ions. It is therefore necessary to stabilise H+ concentration in vitro by adding suitable buffer to the medium. A buffer keeps pH of solution constant by takingup protons that are released during reactions, or by releasing protons when they are consumed. Acetate salt and tris(hydroxymethyl)aminomethane, hydrocloric acid are also used as an buffer in present invention in order to maintain the pH of solution between 4 and 8 to stabilze the ribonucleic acidmolecules. Therefore one of the embodiments of present invention describes the use of water miscible buffer MES/MOPs along with ion sequester and acetate ions.

Mostly, all the RNases are proteins in nature and hence are minorly soluble in water. The effects of salts such as sodium chloride on increasing the solubility of proteins is often referred to as salting in.
When low concentrations of salt is added to a protein solution, the solubility increases as salt molecules stabilize protein molecules by decreasing the electrostatic energy between the protein molecules which increase the solubility of proteins. When the ionic strength of a protein solution is increased by adding salt, the solubility decreases, and protein precipitates, as salt molecules compete with the protein molecules in binding with water.This phenomena is addressed as salting out. Some salts, have general effects on solvent structure that lead to decreased protein solubility and salting out. In this case, the protein molecules tend to associate with each other because protein-protein interactions become energetically more favorable than protein-solvent interaction. Proteins have characteristic salting out points, and these are used in protein separations in crude extracts.The most effective region of salting out is at the isoelectric point of the protein because all proteins exhibit minimum solubility in solutions of constant ionic strength at their isoelectric points. The reagent is capable of protection of ribonucleic acidby various mechanism including, hydration disequilibrium, protein coagulation, disruption of catalytic sites, formation of microspheres, sequestring of cations. The method and reagents disclosed in following specification relates to the field of molecular biology, cell biology and various branches of applied biology.
The ribonucleic acid preserved by present invention can be used for Gene Expression studies, cDNA library preparation, EST-Seq, SAGE-Seq, Gene mining, RNA-Seq/Transcriptome analysis on Next generation sequencing platforms like Genetic analyzer 3730XL, Roche454, SOLiD, illumina, Ion Torrent, Proton, PacBio but not limited to these mentioned platforms.
BRIEF DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention will be further described in the Examples that follow. These
Examples are for illustration purposes only and not intended to limit the invention in any
way. In the Examples reference is made to the following figures.
EXAMPLE 1
Determination of Intactness of RIBONUCLEIC ACID
Samples were immersed in tmsRNA Stabilizer Solution and ribonucleic acid was isolated
from these samples and intactness of isolated ribonucleic acid was checked against

samples that were quick freezen in liquid nitrogen- When degradation of ribonucleic acid takes place, the 28S breaks down at a rapid speed than 18S. Ribonucleic acid quality is mainly determined by sharpness of these bands and ratio of 28S to 18S.Agarose gel electrophoresis is primarily used to determine the intactness of ribonucleic acid (procedure described in Sambrook,2001), denaturing agarose gel containing MOPS and Formaldehyde provides better resoltion of ribonucleic acid bands compared to normal agarose gel. More specifically, the quality of ribonucleic acid is analyzed by capillary electrophoresis system (Bioanalyzer 2100, Agiler|t Technologies), it enables precise measurement of relative areas of 18s and 28s rRNA peaks. According to the inventor, good quality ribonucleic acid preparations should have ribonucleic acid Integrity Number(RIN) greater than 6.5, this figure is variable with the sample origin. Partially degraded, mentioned in this specification refers to ribonucleic acid possissing RIN number 4-5 and ratio of 28S:18S is about 1:1 but bands are intact and distinct. Mostly degraded , mentioned in this specification refers to ribonucleic acid possessing RIN number <4 and ratio of 28S:18S is lower than 1:1, 28S band is blurred but still visible. Completely degraded, mentioned in this specification refers to ribonucleic acid showing RIN number less than 1 and ribonucleic acid present is in a low molecular weight smear below normal position of 5.5S RNA.
EXAMPLE 2
A. Method of preserving tissues in ribonucleic acid protectant media
Plant tissues from Rice, Wheat, Jute, Mango, Jasmicum, Cucumis, Pigeonpea, Tinospora, Giloe, Fenugreek, Prosophis, Bamboo, Andrographis, Brahmi, White Musli, Millet, and Bitterguard etc has been stored in tmsRNA stabilizer solution so that it can effectively penetrate inside the tissue and act on each cell for preservation of ribonucleic acid inside cell. Also, plant tissues was dipped totally in trnsRNA stabilizer solution such that tissue remains completely submerged. Samples was stored in at 25°C, 4°C and -20°C for several days to months. The time points for the study was 2d, 7d, 15d, 30days.. For longer preservation, sample to be kept either at -20°C or tissues alongwith tmsRNA stabilizer solution has to be freezen at -80°C. At above mentioned time interval, samples had been harvested and analysed for morpho/ogical changes as well as the effctes of tmsRNA stabilizer solution on ribonucleic acid stability using bioanalyzer were studied.
B. Process for Isolating ribonucleic acid
a. RNA isolation from Plant tissue Using XcelGen Method :

100-200 mg of plant tissues like leaf, stem, root, fruit, Infurescence, imature/mature seed stored frozen as well as stored in tmsRNA stabilizer solution were ground to fine powder using pre-chilled mortar and pestle. Powdered sample was transferred in 1 ml of extraction buffer containing 100 ul plantaid solution. Half volume of absolute ethanol was added to the flow-through by transferring the solution into a RNA column and centrifuged. 500 ul of buffer RB was added to the column and centrifuged. For washing, 500 ul of RNA wash buffer was added to the column and centrifuged. Elution was carried out in 30 ul of RNase- free water was added to the column and centrifuge at 13,000 rpm for 1 minute and isolated RNA was used to analyse the integriety on RNA6000 picochip or stored at -80°C for later use.
b. RNA isolation from bacterial cell pellet using XcelGen Bacterial RNA
isolation Kit
Aspergillus sp of fungus has been grown in PDB media at 37°C for three to five days and mycelia was harvested and stored in tmsRNA stabilizer solution at 0-37°C, 4°C and -20°C for months and sample was harvested and analysed for the effctes of tmsRNA stabilizer solution on RNA stability using bioanalyzer. Resuspend the tmsRNA stabilizer solution stored 100 mg fungal tissue in a 2 ml tube and grind using a rotor starter. Transfer 10 volume (1 ml) Buffer RLYAmercaptoethanol and 1 volume (100ul) Fungalaid to the tube containing the fungal tissue immediately. Transfer the cleared lysate to a DNA Clearance column and centrifuged. Add 0.5 volume 100% ethanol to the lysate. Transfer the solution into a RNA column and centrifuged at 13,000 rpm for 1 min. Add 500 ul Bu-er RB to the column and centrifuge at 13,000 rpm for 30s and discard the flow-through. Add 500 ul RNA Wash Bu-er to the column and centrifuge at 13,000 rpm for 1 min. Discard the flow-through. Add another 500 ul RNA Wash Bu-er to the column and centrifuge at 13,000 rpm for 30 s. Elution was carried out in 30 ul of RNase- free water was added to the column and centrifuge at 13,000 rpm for 1 minute and isolated RNA was used to analyse the integriety on RNA6000 picochip or stored at -80°C for later use.
c. RNA isolation from Fungal pelleted cells using XcelGen Fungal RNA
isolation Kit
Hallobacillus sp., Bacillus sp., E. coli sp., Klebsiella sp., Micrococus sp., has been grown in nutrient media broth at 37°C for 14-16 hours and cells were harvested and stored in tmsRNA stabilizer solution at 0-37°C, 4°C and -20°C for months and sample was harvested and analysed for the effctes of tmsRNA stabilizer solution on RNA stability using bioanalyzer. Resuspend the tmsRNA stabilizer solution stored pellet in 100 pi freshly

prepared TE Buffer(10mM,Tris-HCL,PH 8.0;1mM EDTA.PH 8.0) or Elution Buffer (10mM Tris-HCL, PH 8.5) containing lysozyme. (Use 3 mg lysozyme per 1 ml TE Buffer for Gram-positive bacteria and 0.4 mg/ml lysozyme for Gram-negative bacteria). Mix by tapping gently. Incubate the resuspended pellet at room temperature for 5-10 min for Grampositive bacteria, or 3-5 min for Gram-negative bacteria. Add 400 ul Buffer LY. Mix gently. Transfer the cleared lysate to a DNA Clearance column pre-inserted in a 2 ml Collection Tube. Centrifuge at 13,000 rpm for 2 min. Discard the DNA Clearance column and save the flow-through. Note: This step is for genomic DNA removal, it is not necessary. Transfer flow-through to a new RNase-free tube. Add 0.5 volume 100% ethanol to the lysate (For example: 250 ul 100% ethanol for 500 ul lysate). Ensure that P-mercaptoethanol has been added before use. Transfer the solution into the binding column and centrifuge at 13,000 rpm for 1 min. Discard the collection tube with the flow-through and put the column back to a new collection tube. Add 500 ul Buffer RB to the column and centrifuge at 13,000 rpm for 30s. Discard the flow-through. Add another 500 pi RNA Wash Buffer to the column and centrifuge at 13,000 rpm for 30s. Discard the flow-through. Ensure that ethanol has been added to RNA Wash Buffer before use. Add another 500 pi RNA Wash Buffer to the column and centrifuge at 13,000 rpm for 30 s. Discard the flow-through and collection tube, put the column into a new collection tube. Centrifuge the column at 13,000 rpm, with the lid open, for another 1 min. It is critical to remove residual ethanol for optimal elution. Place the column to a RNase-free 1.5 ml tube, add 50-100 ul DEPC-treated ddH20 to the column and centrifuge at 13,000 rpm for 2 min. The RNA is in the flow-through liquid, isolated RNA was used to analyse the integriety on RNA6000 picochip or stored at -80°C for later use:
EXAMPLE 3
Presentation of tissues in stabilizer solutions:
In this example, the stabilizer solution comprises of 4% polyethylene glycol 200, which was dissolved in water and added in quartinary salt solution. 25mM polyamino carboxylic acid in 25mM citrate solution and 5% v/v polyol has been added in the solution. The pH of the solution was adjusted to 6.67 using acid, the solution was filtered through 0.45 urn filter, solution was trasferred to 1.5ml tubes to store the tissues at three different temperatures in two replicates, plant tissues, fungal cell pellet, bacterial cell pellet, insect tissue were submerged into the solution. The tissues submerges in this composition were then transferred to 3 different temperatures - (1) 25°C (2) 4°C and (3) -20°C. Morphology of the plant tissues was recorded for seven days, leaf tissue morphology changes was observed after 7days of incubation at 25°C, 4°C and -20°C as described in Fig.1 a, b and c and Fig.2

a,b and c, more detrioration was observed at 25°C in comparision to low temparature. Total RNA isolated from plant leaf after immersing in tmsRNA stabilizer from 2day and 7th day stored at low temparature. The RNA was resolved on denaturing agarose gel and found the degradation of RNA molecules from 2nd day to 7th day storage as described in Fig3A and B. The composition of this solution was able to protect the RNA molecule in other part of plant tissues as well as in bacterial and fungal cell pellet for several weeks at ultra low temprature.
EXAMPLE 4
Dtermining the effects of morphalinopropanesulphonic acid
A similar composition to RNA protection solution was prepared containing 200 mM, 2-Amino-2-hydroxymethyl-1,3-propanedioi hydrochloride, 10mM KH2P04 250mM 3-(N-morpholino)propanesulfonic acid, 25mM polyamino carboxylic acid in quartinary salt solution, poly-ol 5%, Isopropyl alcohol 5%, polyethylene glycol <10%. The buffering of the solution was adjusted to pH 6.67 using acid, the solution was filtered through 0.45 urn filter, solution was trasferred to 1.5ml tubes to store the tissues at three different temperatures in three replicates, plant tissues, fungal cell pellet, bacterial cell pellet, insect tissue were submerged into the solution. The tissues submerges in this composition were then transferred to 3 different temperatures - (1) 25°C (2) 4°C and (3) -20°C. Morphology of the plant tissues was recorded for seven days. In case of plant leaf tissue decoloration and degradation was observed after 6th day onward upon incubation at 25°C as described in Fig.4 (Fig4.1 to 4.3). The RNA was isolated from all the samples stored using method described elswhere in the document. Isolated RNA was analyzed on denaturing agarose gel as well as on RNA chip, which confers the degradation of RNA in sample stored at 25°C after 7th day onward as evident by morphologocal changes, while intact RNA was observed in rest of the samples stored at ultra low temprature. Therefore the composition of this example was able to protect the ribonucleic acidmolecule molecule in plant tissues as weil as in bacterial and fungal cell pellet for several days to several weeks at ultra low temprature.
EXAMPLE 5
Determining the effect of 3-(N-morpholino)propanesulfonic acid and
polyethylene glycol in ribonucleicacid preservation
A composition of this example is to determine the effect of the polyethyleneglycol effects on RNA protection, consist of 100 mM 2-Amino-2-hydroxymethyl-1,3-propanediol

hydrochloride, 10mM KH2P04, 200mM 3-(N-morpholino)propanesulfonic acid, poly-ol 2%, 25mM polyamino carboxylic acid in quartinary diazanium salt solution. The buffering of the solution was adjusted to pH 6.67 using acid, the solution was filtered through 0.45 urn filter, solution was trasferred to 1.5ml tubes to store the tissues at three different temperatures in three replicates. The plant leaf tissue was stored 3 different temperatures - (1) 25°C (2) 4°C and (3) -20°C. The precipitation was observed at the bottom of the vial indicating after 24 hrs in samples stored at 4°C as well as -20°C, as described in Fig.5 (Fig5.1, 5.2 and 5.3). Microscopic observation of leaf surface shows cell morphology disintegration after 10th day of incubation at 25°C, while sample store at low temparature has some what intcat morphology. RNA isolated from samples incubated at 25°C, shows degradation, in comparison to low temparature storage, on denaturing agarose gel.
EXAMPLE 6
Dtermining the effects of morphalino acid, Chelators and polyaminocarboxilic
acid in ribonucleicacid preservation
The composition of ribonucleic acid stabilizing solution was prepared containing 200 mM 2-Amino-2-hydroxymethyl-1,3-propanediol hydrochloride, concentration of 2-(N-morpholino)ethanesulfonic acid and 3-(N-morpholino)propanesulfonic acid are ranges upto 97.60 gram per liter and 104.6 gramper liter respectively, poly-ol 2% in 25mM sodium citrate, 25mM polyamino carboxylic acid in quartinary diazanium salt solution. The buffering of the solution was adjusted to pH 6.67 using acid, the solution was filtered through 0.45 urn filter, solution was trasferred to tubes to store the tissues at three different temperatures in three replicates. Plant tissues, Bacillus bacterial cell pellet and Aspergillus fungal mycelial cell pellet, were submerged in this composition were then transferred to 3 different temperatures - (1) 25°C (2) 4°C and (3) -20°C. The time point for the study was 2d, 7d, and 15d. Also rice and wheat leaf tissue has been stored for extended period of storage for 3 months and 6 months. Morphology of the plant tissues and RNA quality was recorded for all time points, the Fig 6.1, 6.2 indicate the tissue quality at 0 day and 15 days, while rice and wheat leaf morphology was also intact on extended storage at -20°C for 3 months and 6 month respectively. RNA isolation was carried out using the method described in above examples for all time points and samples were analyzed on RNA6000 picochip as per as per instrutions mentioned in Agilent's manual. The results are smarized in table 1, indicating that RNA integriety is maintained in condition described above as explained in Fig.7 to Fig11 for plant, bacteria as well as for fungus cells, indicates that the composition is enable to preserve the morphology and ribonucleic acid when stored post harvest.

TABLE 1


DRAWINGS

Fig.1-day2

Fig.2-day7

A B Fig.3

BRIEF DESCRIPTION OF THE DRAWING: Fig.1
The plant leaf has been stored in the composition described in exaple 3 for at 25°C, 4°C and -20°C temparature; a, b and c) Thre was no change in morphology of leaves was observed after 2 days storage at either 25°C, 4°C and -20°C. Fig.2
The plant leaf has been stored in the composition described in exaple 3 for at 25°C, 4°C and -20°C temparature; a, b and c) leaf tissue morphology changes was observed after 7days of incubation at 25°C, 4°C and -20°C, more detrioration was observed at 25°C in compariston to low temparature.
Fig.3
Total RNA isolated from plant leaf after immersing in tmsRNA stabilizer composition described in example 3 from 2day and 7th day stored at low temparature. The RNA was resolved on denaturing agarose gel and found the degradation of RNA molecules from 2nd day to 7th day storage.
Fig.4
Plant leaf tissue stored in composition described in exmple 4, were examined by microscopy for 2nd day ( Fig 4.1), 7th day (Fig 4.2) and 15th day ( Fig. 4.3) stored at 25°C, indicating the detrioration of the morphology from 7th day onward.
Fig.5
Plant leaf tissue stored in composition described in exmple 5, were examined by

microscopy for 2nd day ( Fig 5.1), 7th day (Fig P-2) and 15th day { Fig. 5.3) stored at 25°C, indicating the detrioration of the morphology at 15 day onward.
Fig.6
Plant leaf tissue stored in composition described in exmple 6, were examined by microscopy for 2nd day, and 30th day stored at 25°C as depicted in Fig 6.1 and Fig 6.2 respectively, indicating intect morphology. The rice and wheat samples stored in this composition are having intect morphology til 3 months and 6 months respectively at -20°C as described in Fig 6.3 and 6.4.
Fig. 7A, 7B and 7C
various temparature like; 25°C, 4°C and -20°C- The RNA was resolved on RNA picochip and intact RNA has been observed.
Fig. 8A, SB and 8C
Total RNA isolated from stem of a plant, after immersing in RNA stabilizer media for 7 day at various temparature like; 25°C, 4°C and -20°c- The RNA was resolved on RNA picochip and intact RNA has been observed. Fig. 9A, 9B and 9C
Total RNA isolated from lesf of a plant, after immersing in RNA stabilizer media for 7 day at various temparature like; 25°C, 4°C and -20°C- Tne RNA was resolved on RNA picochip and intact RNA has been observed. Fig. 10A, 10B, &10C
Total RNA isolated from Bacillus bacterial cell Pellet after immersing in RNA stabilizer media for 7 day at various temparature like; 25°C, 4°C and -20°C. The RNA was resolved on RNA picochip and intact RNA has been observed-Fig.11A, 11B&11C
Total RNA isolated from Aspergillus fungal mycelial pellet, after immersing in RNA stabilizer media for 7 day at various temparature like; 25°C, 4°C and -20°C. The RNA was resolved on RNA picochip and intact RNA has been observed-

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Sidney Altman(Department of Molecular, Cellular and Developmental Biology Yafe
University
New Haven, Connecticut 06520 Leif Kirsebom)
Department of Microbiology(Biomedical Center Uppsala University Uppsala S-751 23,
Sweden)
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CLAIMS:
What is claimed is:
1. A composition for stabilizer solution for preservation of various type of ribonucleic acidin completely submerged biological samples consiting of harvested prokaryotic cells, eukaryotic cells, tissues from endonuclease and exonuclease degradation.
2. The composition of claim 1, wherein the stabilizing solution consist of hydrophilic ion molecules to disrupt the hydration sphere around the catalytic active proteins.
3. The method in claiml, wherein stabilizing agent are 2-(N-morpholino)ethanesulfonic acid and 3-(N-morpholino)propanesulfonic acid in sodium citrate or the derivatives of the same.
4. The method in claim1, wherein stabilizing agent concentration of 2-(N-morpholino)ethanesulfonic acid and 3-(N-morpho.lino)propanesulfonic acid are ranges upto 97.60 gram per liter and 104.6 gramper liter respectively.
5. The method in claiml, wherein stabilizer solution comprises of polyamino carboxylic acid which is also a conjugate base, possessing ability to sequester divalent and tetravalent cations such as Mg+2, Ca+2, Fe+2 and Fe+3.
6. The method in claiml, wherein polyamino carboxylic acid concentration ranges upto 19.02 gram per liter.
7. The method of claiml, wherein the stabilizing solution comprising of tris(hydroxymethyl)aminomethane salt or quaternary diazanium salt.
8. The method of claiml, wherein the stabilizing solution comprising of tris(hydroxymethyl)aminomethane compound concentration ranges from 12.14 gram per liter to 121.14 gm per liter.
9. The method of claiml, wherein the stabilizing solution comprising of dibasic phosphate ions having concentration of 100 gram per liter to 200 gram per liter.

10The composition of claim 1, wherein the stabilizing solution comprising of polyols with concentration of 0.1 percent to 20 percent.