RU2322058C2 - Method for preserving biological sample providing safety of nucleic acids - Google Patents

Abstract

FIELD: medicine, biotechnology, biochemistry.

SUBSTANCE: invention relates to methods for preparing for analysis of nucleic acids isolated from biological material used mainly for detection of specific RNA and DNA molecules in biological material. Preserving method involves preparing a lysate by mixing biological material with a preserving solution that contains guanidine compound in the amount providing the essentially complete denaturation of nucleases in a sample to be analyzed, namely with a reducing agent of protein S-S-bonds taken in the amount providing the essentially complete reduction of S-S-bonds in lysate proteins, with a strong detergent not forming insoluble compound with guanidine and taken in the amount providing formation of micelles in lysate with at least one chelating agent for ions of two- and trivalent metals and taken in the amount providing its molar concentration in lysate that is known to be higher as the compare maximal molar concentration of ions of two- and trivalent metals, and by a buffer maintaining pH value of lysate in the range from 6 to 8. Method provides safety of nucleic acids in prolonged storage of samples in a not frozen state.

EFFECT: improved preserving method.

13 cl, 1 dwg, 3 ex

Description

Technical field

The invention relates to the field of molecular biology and biotechnology, in particular to a method for the conservation of biological samples.

State of the art

Isolation of intact nucleic acids - DNA and / or RNA - from biological samples is a prerequisite for many types of experiments and analytical studies, including cloning of genetic material and various types of molecular diagnostics based on the multiplication of nucleic acids, such as PCR (polymerase chain reaction) or isothermal propagation methods considered, for example, in [1, 2]. Often a lot of time passes between the collection of biological material and the release of nucleic acids from it; Often the collected material must be transported a considerable distance at a relatively high temperature. It is also convenient to collect a series of samples for some time, and then process all samples at the same time. In all these and in many other cases, the samples must be stored for some, sometimes long, time, and it is very important to ensure such storage conditions that the integrity (intactness) of nucleic acids is preserved.

It is important that storage conditions are compatible with subsequent use of nucleic acids. For example, drugs (for example, vanadate-ribonucleoside complexes), which may subsequently lead to inhibition of PCR, should not be introduced into the sample. It is also important that the method for storing samples does not require the use of expensive and bulky equipment (for example, low-temperature freezers), it is cheap and simple.

To date, many methods for storing biological material have been proposed. The most common is frozen storage. To prevent degradation of nucleic acids, they try to freeze samples very quickly (for example, in liquid nitrogen) and store them at a very low temperature (for example, at -80 ° C). In addition to the fact that this method is too expensive and unavailable for many clinical laboratories, it also does not ensure the complete preservation of nucleic acids, since during thawing due to the destruction of intracellular structures, nucleases are released that quickly hydrolyze DNA and, especially, RNA. To shorten the thaw time and reduce the degradation of nucleic acids, the sample is ground into powder in a frozen state. However, this method is very time-consuming and unsuitable for diagnostic purposes, as it is associated with a high risk of mutual contamination of the samples.

It was proposed to keep the samples fixed in a mixture of 5% acetic acid, 95% ethanol and ribonuclease inhibitors [3]. In addition to the fact that this method does not ensure complete preservation of RNA [4], it is also associated with DNA degradation (which is susceptible to hydrolysis in an acidic environment). Other variants of alcohol solutions, mixtures of alcohols with acetone (for example, considered in the publication [4]), and mixtures of methanol with polyethylene glycol [5] were also proposed. However, in addition to the fact that storage in such solutions does not ensure the safety of RNA at room temperature [4], intense degradation of nucleic acids can occur due to reactivation of nucleases at the time of replacement of alcohols with aqueous solutions in which nucleic acids are isolated. Similar problems arise when using substances that cause precipitation of RNA from aqueous solutions, such as triphenylmethane dyes or cobalt salts [6]; in addition, in this case, cobalt dyes and ions can inhibit subsequent PCR.

It was also proposed to stabilize RNA in solution with a strong detergent lithium dodecyl sulfate [7]. We tried to use this method, but found that when processing samples with a high content of ribonucleases, such as whole blood, RNA degrades in the first seconds after the addition of dodecyl sulfate. The fact is that dodecyl sulfate significantly increases the availability of nucleases to RNA, destroying subcellular structures and RNA-protein complexes, than inactivates nucleases. In addition, inactivation of ribonucleases by dodecyl sulfate is significantly reversible.

The strongest of the known denaturants of ribonucleases is guanidine thiocyanate, which is widely used in the isolation of RNA [8]. However, there are no known examples of the successful use of this substance for long-term storage of biological material. Moreover, the author of the patent [4] demonstrates that in the presence of guanidine thiocyanate RNA almost completely degrades within 12 hours at + 4 ° C, indicating the possibility that guanidine itself is a catalyst for RNA hydrolysis. The same author suggests using salting out of samples with high salt concentrations (preferably ammonium sulfate), sufficient for RNA precipitation. Although the author of the patent [4] gives positive examples of the use of salting out for the preservation of tissue pieces, he briefly mentions the possibility of using this method for the preservation of cell suspensions, in particular blood. When checking this method of blood preservation, it was found that (1) when the concentration of ammonium sulfate recommended by the author of the patent [4], precipitated precipitate does not form. Instead, a jelly-like mass forms, which only slightly compacts during centrifugation. Nucleic acids could not be isolated from this mass. When guanidine thiocyanate was added, it was dissolved, but nucleic acids could not be precipitated from the resulting solution by methods commonly used to precipitate RNA and DNA from guanidine thiocyanate lysates in the absence of ammonium sulfate, for example, by adding isopropanol.

As a prototype of the present invention can be considered a patent [4], as well as related patents [9, 10].

We doubted that guanidine thiocyanate itself could cause RNA degradation and suggested that the fast RNA degradation described in the patent [4] was caused by some other factors. In the case of biological samples, especially animal tissues, there can be many such factors. First of all, these are ribonucleases, ions of divalent and trivalent metals and peroxides, which are a source of free radicals. Unfortunately, the author of the patent [4] does not provide a detailed description of the experiment, but merely indicates that “the samples were placed ... in a 4”, based on 4M guanidine thiocyanate at 4 ° C for 12 hours. molar guanid-inium isothiocyanate (GITC) based RNA extraction solution (lanes 1, 3, and 5) at 4 ° C for 12 hours "- see signature to FIG. 2 of patent [4]). Therefore, it is unclear what other substances and in what concentrations were present in the solution, and also under what conditions (pH, illumination) the experiment was conducted.

During the experiments it was found that with the correct composition of the preservation solution prepared on the basis of guanidine thiocyanate, RNA and DNA remain intact at + 4 ° C for at least 2 weeks. Moreover, in such a solution, RNA remains in a substantially intact state at room temperature for up to 3 days. It was also found that the safety of RNA at room temperature can be extended to at least 2 weeks if nucleic acids are planted with a water-soluble organic solvent (drawing). Thus, it was demonstrated that guanidine thiocyanate is not a factor causing RNA degradation.

The aim of this invention is to increase the safety of RNA and DNA for a long time, including at relatively high temperatures. Another objective is to develop a method compatible with the subsequent isolation of pure nucleic acids from canned samples and various analysis methods, including PCR and other nucleic acid multiplication systems.

SUMMARY OF THE INVENTION

These goals are solved by the fact that the proposed method of preservation of biological samples, which involves obtaining a lysate by mixing the biological material with a preservation solution containing the guanidine compound in an amount sufficient to substantially completely denature the nucleases contained in the sample, the preservation solution also containing the following components that prevent degradation nucleic acids: (1) a reducing agent for protein SS-bonds in an amount providing substantially complete restoration of SS-bonds her lysate proteins; (2) a strong detergent that does not form an insoluble compound with guanidine in an amount sufficient to form micelles in the lysate; (3) at least one complex of ions of divalent and trivalent metals in an amount providing its molar concentration in the lysate is obviously higher than the expected maximum molar concentration of ions of divalent and trivalent metals; (4) a buffer that maintains the pH of the lysate in the range of 6 to 8.

The proposed method of conservation ensures the storage of samples for a long time, as well as the safe transportation of samples at ambient temperature. Finally, the proposed conservation method is compatible with the subsequent nucleic acid isolation procedure.

List of drawings

The invention is illustrated in the drawing:

the use of the method for the preservation of blood samples, especially rich in ribonuclease and iron ions, where: (A) - whole blood preparations were analyzed by electrophoresis in SDS page, followed by staining with ethidium bromide, detecting mainly DNA, (B) - data of autoradiography obtained using phosphoimager "Cyclon".

Description of the invention

Based on the research results, a method for the conservation of biological samples is proposed, which ensures the substantial safety of RNA and DNA during storage of samples until nucleic acids are isolated. The essence of the invention lies in the fact that biological samples are mixed with a preservation solution, which (1) contains, in addition to guanidine thiocyanate, a number of components that prevent the degradation of nucleic acids under the influence of factors present in biological samples or formed during their storage and (2) has the pH value at which the lowest rate of hydrolysis of DNA and RNA and the lowest rate of formation of free radicals are observed. If necessary, the sample is homogenized during the addition of the preservation solution. Further, the sample processed in this way will be called a lysate.

According to the invention, the preservation solution contains the following components that prevent the degradation of nucleic acids.

(1) A guanidine compound, preferably guanidine thiocyanate, but it is also possible to use chloride or other salts of guanidine, as well as mixtures of different guanidine compounds. The final concentration of the guanidine compound in the lysate should ensure, taking into account the presence of other components of the preservation solution, substantially complete denaturation of proteins, primarily complete denaturation of ribonucleases and deoxyribonucleases (together, nucleases). In the case of guanidine thiocyanate, the preferred concentration in the lysate is 4 M, in the case of guanidine chloride, 6 M.

(2) A compound that restores S-S bonds in proteins, with the goal of substantially complete inactivation of nucleases, taking into account the presence of other components of the preservation solution. The preferred compound is 2-mercaptoethanol (β-mercaptoethanol) due to its relative cheapness, but other reducing agents such as dithiothreitol, thiourea and glutathione can also be used. The final concentration of the reducing agent in the lysate should provide, taking into account the presence of other components of the preservation solution, a substantially complete restoration of S-S bonds in the lysate proteins. The preferred concentration of 2-mercaptoethanol in the lysate is from 1 to 2% (by volume). The preferred concentration of other reducing agents may be different. For example, the reduction potential of dithiothreitol is higher than 2-mercaptoethanol; therefore, it is preferable to use it 4-6 times lower concentration.

(3) Strong detergent that does not form an insoluble compound with guanidine when they are combined in a preservative solution at its storage temperature (which is preferably room temperature). The detergent promotes complete inactivation of nucleases, and also improves lysis of the cells and tissues of the sample. Sarcosyl (N-dodecyl sarcosine or N-lauryl sarcosine) is the preferred detergent, but other detergents such as the polyethylene glycol ethers known under the Triton trademark (for example, Triton X-100 tert-octylphenyl polyethylene glycol ether) and polyoxyethylene sorbitan can be used. trademark Tween (for example, Tween 20 - polyoxyethylene sorbitan monolaurate), or their chemical analogues. For example, dodecyl sulfate, which forms an insoluble compound with guanidine at room temperature, is not suitable. The concentration of detergent in the lysate should be high enough to form micelles. The preferred concentration of sarcosyl in the lysate is 0.5%.

(4) At least one complexon (chelator) of divalent and trivalent metal ions, which are effective catalysts for RNA hydrolysis [11]. Of particular risk are ions Fe ²⁺ and Fe ^3+, are present in animal tissues in large amounts in the composition of hemoglobin released during denaturation. The preferred complexone is EDTA (ethylene diamine-N, N, N ', N'-tetraacetate), the binding constants of which with iron ions are ≈10 ^-14 M ^-1 (Fe ²⁺ ) and ≈10 ^-24 M ^-1 (Fe ^{3 +} ) [12]. Other complexones such as EGTA (ethylene glycol bis [2-aminoethyl] -N, N, N ′, N′-tetraacetate), 8-hydroxyquinoline and 1,10-phenanthroline can also be used. The molar concentration of complexon should be obviously higher than the expected maximum molar concentration of ions of divalent and trivalent metals. The preferred concentration of EDTA in the lysate is 10-20 mM.

(5) A buffer that maintains a pH in the range from 6 to 8. At pH> 8, alkaline hydrolysis of RNA is possible, and at pH <6, acid apurination of DNA is possible with subsequent degradation of its sugar phosphate backbone [13]. In addition, with a decrease in pH from 6 to 4, the so-called Fenton reaction is accelerated many times [14], in which free radicals are formed with the participation of Fe ²⁺ from peroxides that can be present in tissues or form under the influence of light in the presence of dissolved oxygen. first of all, hydroxyl radicals causing nonhydrolytic decomposition of both RNA [15] and DNA [16]. As a rule, biological lysates have an acidic reaction, therefore, the preferred buffer is a citric acid salt, for example, trisubstituted sodium citrate, which effectively maintains the pH above 6, while being an effective complex of Fe ³⁺ ions [12]. The preferred concentration of buffer in the lysate is 20-30 mM, but higher concentrations can be used if necessary.

It should be noted that, in addition to the buffer, Fenton’s reactions are also hindered by other components of the preservative solution, such as reducing agents of SS bonds and complexons of ions of divalent and trivalent metals [14], and in order to avoid the formation of peroxides, the lysate should be stored in a dark place and / or in a lightproof bowl.

The preferred composition of the preservation solution and the preferred method for preserving the biological sample are shown in Example 1, and the result of its use for the preservation of blood samples, especially rich in ribonuclease and iron ions, is shown in the drawing.

According to the invention, the safety of RNA in the lysate can be further increased by subsequent mixing of the lysate with a water-soluble organic solvent capable of causing precipitation of nucleic acids. Many organic solvents possess such properties, for example, low molecular weight alcohols (such as methanol, ethanol, and isopropanol), acetone, polyethylene glycol, and dimethyl sulfoxide [17]. Isopropanol is preferred according to the invention. The amount of added organic solvent should ensure substantially complete precipitation of nucleic acids, but not be excessive so as not to cause excessive precipitation of proteins. The preferred amount of isopropanol is 2 volumes per 1 volume of lysate; the optimal amount of other solvents may be different and should be established in preliminary experiments. A preferred method for preserving a biological sample using isopropanol is shown in Example 2, and the result of its use for preserving blood samples is shown in the drawing.

It can be noted that as the lysate is stored in the presence of isopropanol, the amount of DNA released from the lysate decreases according to the method described in Example 3. However, this does not noticeably increase the content of low molecular weight fragments, as would be expected during DNA degradation. The most likely reason for the decrease in yield is DNA loss caused by a deterioration in its solubility during storage in the presence of isopropanol. Probably, during storage, dense DNA-protein aggregates are formed, which are lost during phenolic extraction, passing into the organic phase. Therefore, if it is desirable to isolate DNA from such a sample with maximum yield, the sample should be treated with proteinase K (or another similar protease) before phenol extraction, preferably in the presence of urea and detergent, as described, for example, in [18].

The lysate obtained by mixing a biological sample with a preservative solution according to the invention is fully compatible with most of the widely used nucleic acid isolation procedures. The chemical composition of this lysate is close to the lysate obtained by various methods of nucleic acid isolation, including the isolation of RNA by extraction with acid phenol [19] and the isolation of RNA and DNA by adsorption on finely dispersed silicate or glass carriers [20] used , for example, in a NucliSens extractor (Organon Teknika, Netherlands). In fact, the process of preservation of the sample replaces the stage of obtaining the lysate provided for by these methods.

The proposed method is illustrated by the following examples of its implementation.

Example 1. Preservation of blood and bone marrow samples.

The following is a procedure calculated to process 0.15 ml of blood. Similar results are obtained with the conservation of 0.15 ml of other cell suspensions, for example, bone marrow. When preserving a sample of a different volume, the volumes of the added solutions should be proportionally changed. When preserving hard tissues, they should preferably be homogenized in a preservation solution in any way possible (in a blender, Potter homogenizer, ultrasonic homogenizer, etc.).

0.15 ml of freshly collected blood was poured into a test tube into which a preservation solution (0.3 ml) was added in advance, and stirred rapidly. The preservation solution had the following composition: 6 M guanidine thiocyanate, 37.5 mM sodium citrate (pH 7.5), 0.75% sarcosyl, 20 mM EDTA, 2% 2-mercaptoethanol (final concentrations: 4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 13 mM EDTA, 1.3% 2-mercaptoethanol). The resulting lysate was stored at different temperature conditions [room temperature ("rt"), as well as + 4 ° C and -20 ° C] for 3, 7 and 14 days. Then, nucleic acids were isolated by the method described in example 3, starting from point 2, and the part of the obtained preparation, corresponding to 20 μl of blood, was analyzed by electrophoresis in SDS page. The results are presented in the drawing. DNA preservation, which amounts to 90% of the mass of the isolated preparation, was monitored by ethidium bromide staining (A), and RNA preservation was monitored by the safety of internal control, ³² P-labeled RNA phage QP 3821 nt long, which was added to the blood together with preserving solution and tracked using autoradiography (B). Samples obtained by preservation by the method described in this example have odd numbers. Lanes “K” contain control samples from which nucleic acids were isolated immediately after the addition of the preservation solution, and tracks “M” contained sets of DNA size markers.

The above preservative solution is stable at temperatures above + 20 ° C and can be stored (preferably in the dark) for at least two months without significant deterioration. However, when the temperature drops below + 20 ° C, crystals can precipitate from the solution, which can be dissolved by heating. Therefore, if it is expected that the ambient temperature may drop below + 20 ° C, then the preservative solution is preferably diluted, respectively increasing its volume added to the biological sample, so that the indicated final concentrations of reagents are observed. For example, a preservative solution diluted 1.125 times can be stored with a prolonged decrease in temperature to + 15 ° C, while 3 volumes of preservative solution should be added per 1 volume of sample.

Example 2. Preservation of blood samples, including the precipitation of nucleic acids using an organic solvent.

0.15 ml of freshly collected blood was mixed with 0.3 ml of preservation solution as described in Example 1, after which 0.9 ml of isopropanol was added and mixed vigorously (i.e., 2 volumes of isopropanol per 1 volume of lysate were added). The resulting suspension was stored at different temperature conditions [room temperature ("rt"), as well as + 4 ° C and -20 ° C] for 3, 7 and 14 days. Then, nucleic acids were isolated by the method described in example 3, starting from step 3 (after having subjected the sample to centrifugation, as described in step 2), and the part of the obtained preparation, corresponding to 20 μl of blood, was analyzed by PAGE. The results are presented in the drawing. Samples obtained by preservation by the method described in this example have even numbers. The remaining experimental conditions are the same as in example 1.

If a more diluted preservative solution is used (see Example 1), then the added volume of isopropanol is increased accordingly to maintain a volume ratio of 2: 1 (isopropanol: lysate).

Example 3. Isolation of nucleic acids from whole blood and bone marrow.

All operations were performed in Eppendorf type microcentrifuge tubes, and centrifugation was carried out, unless otherwise indicated, in the cold (4 ° C) in MiniSpin ™ centrifuges (Eppendorf, Germany) at maximum speed (13,400 rpm), using aliquots to isolate 150 μl of whole blood or bone marrow, or the corresponding amount of preparations subjected to preservation as described in examples 1 and 2.

(1) Lysis (this stage is identical to preservation, therefore, it was skipped if nucleic acids were isolated from stored samples). 150 μl of blood or bone marrow was mixed with 300 μl of a lysing solution containing 6 M guanidine thiocyanate, 37.5 mm sodium citrate (pH 7.5), 0.75% sarcosyl, 20 mm EDTA, 2% 2-mercaptoethanol (final concentrations : 4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 13 mM EDTA, 1.3% 2-mercaptoethanol). The solution was thoroughly mixed. Incubated at 0 ° C (in an ice bath) for 5 minutes.

(2) Isopropanol precipitation (this step was skipped if nucleic acids were already precipitated with isopropanol during conservation). 900 l of isopropanol was added to the lysate, and stirred vigorously. Incubated at 0 ° C for 30 minutes and centrifuged for 15 minutes. The supernatant was discarded.

(3) Dissolution of the precipitate. 400 μl of lysis solution diluted with water 1.5 times was added to the precipitate. Final concentrations: 4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 13 mM EDTA, 1.3% 2-mercaptoethanol. The solution was thoroughly mixed.

(4) Precipitation with isopropanol. 800 μl of isopropanol was added, mixed vigorously. Incubated at 0 ° C for 30 minutes and centrifuged for 15 minutes. The supernatant was discarded.

(5) Flushing the nucleic acid precipitate with an alcohol-salt solution. To the precipitate was added 400 μl of a solution containing 165 mM sodium chloride, 110 mM sodium citrate (pH 7.0), 45% (v / v) 96% ethanol. It was stirred at room temperature for 10 minutes on a Reax-Top ™ shaker (Heidolph, Germany) at maximum intensity (2500 rpm), and then centrifuged for 15 minutes. The supernatant was removed.

(6) Dissolution of the precipitate. 500 μl of a solution containing 10 mM Tris HCl (pH 8.0), 1 mM EDTA, 8M urea, 2% SDS, 2% 2-mercaptoethanol was added to the precipitate. Stir at room temperature for 10 minutes on a Reax-Top ™ shaker at maximum intensity.

(7) Phenol Extraction. First, 45 μl of 4 M sodium chloride (up to 300 mM) was added to the solution for subsequent precipitation. Next, 545 μl (one volume) of a phenol: chloroform: isoamyl alcohol mixture (24: 24: 1, by volume) was added (phenol saturated with Tris-HCl buffer, pH 8.0 was used). Stirred at room temperature for 2 minutes on a Reax-Top ™ shaker at maximum intensity. Centrifuged at room temperature for 10 minutes. 400 μl of the aqueous phase was collected. The aqueous phase was washed with 400 μl of chloroform (stirred for 1 min, centrifuged for 1 min), 360 μl of the aqueous phase was taken.

(8) Ethanol precipitation. 1 ml of cold (-20 ° C) 96% ethanol was added to the aqueous phase and stirred vigorously. Incubated at -20 ° C for 20 minutes, then centrifuged for 15 minutes. The supernatant was removed.

(9) Washing the precipitate with alcohol. 400 μl of 75% ethanol was poured onto the precipitate and the tube inner surface was gently washed. Centrifuged for 10 min. The supernatant was discarded, and the precipitate was dried for 10-15 minutes at room temperature.

(10) Dissolution of the precipitate. The precipitate was dissolved in 40 μl of 0.1 mM EDTA, heated for 30 minutes at 60 ° C, and stirred on a Reax-Top ™ shaker at maximum intensity for 3 minutes. After that, it was again heated at 60 ° C for 15 min, then placed in a boiling bath for 3 min (to melt possible aggregates of molecules) and quickly cooled in ice.

Industrial applicability

Based on the results obtained, it can be stated that the method of preservation of biological samples ensures substantial preservation of RNA and DNA during storage of samples until nucleic acids are isolated for a long time, as well as safe transportation of samples at ambient temperature. Finally, the proposed conservation method is compatible with the subsequent nucleic acid isolation procedure.

Information sources

1. Chetverin A.B., Chetverina E.V. The method of propagation of nucleic acids, the method of expression and the environment for their implementation // RF Patent No. 2048522 (1995).

2. Chetverin A.B., Chetverina E.V. Accurate diagnosis using molecular colonies // Molecular. biology. T.36. S.320-327 (2002).

3. Esser C., Gottlinger C., Kremer J., Hundeiker C., Radbruch A. Isolation of full-sized mRNA from ethanol-fixed cells after cellular immunofluorescence staining and fluorescence-activated cell sorting (FACS) // Cytometry. V.21. P. 382-386 (1995).

4. Lader E.S. Methods and reagents for preserving RNA in cell and tissue samples // United States Patent 6204375 (2001).

5. Vincek, V., Nassiri, M., Nadji, M., Morales, A.R. Preservation of RNA and morphology in cells and tissues // United States Patent Application 20030211452 (2003).

6. Kamme F.C., Meurers B.H., Talantov D., Yu J. Preservation of RNA in a biological sample // United States Patent Application 20040259133 (2004).

7. Stefan M., Krehan A.-A., Boecher O., Waschuetza S. Use of lithium dodecyi sulfate for stabilizing RNA in solution, particularly during purification of RNA from cell lysate // Patent DE 10219117 (2003).

8. Chirgwin J.M., Przybyla A.E., MacDonald R.J., Putter W.J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease // Biochemistry. V.18. P.5294-5299 (1979).

9. Lader E.S. Methods and reagents for preserving RNA in cell and tissue samples // PCT publication WO 0006780 (2000).

10. Lader E.S. Methods and reagents for preserving RNA in cell and tissue samples // United States Patent 6528641 (2003).

11. Feig A. L, Uhlenbeck O.C. The role of metal ions in RNA biochemistry // The RNA World, 2nd ed. (Eds. Gesteland R.F., Cech T.R., Atkins J.F.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. P.287-319 (1999).

12. Sillen, L. G., Martell, A.E. Stability Constants of Metal-Ion Complexes. The Chemical Society, London, 1964.

13. Davidson, J. Nucleic Acid Biochemistry. "Mir", Moscow, 1976.

14. Walling, S. Fenton's reagent revisited // Ace. Chem. Res. V.8. P.125-131 (1975).

15. Latham J.A., Cech T.R. Defining the inside and outside of a catalytic RNA molecule // Science. V.245. P.276-282 (1989).

16. Tullius T.D., Dombroski B.A. Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda represser and Cro protein // Proc. Natl. Acad. Sci. U.S.A. V.83. P.5469-5473 (1986).

17. Chomczynski P. Product and process for isolating DNA, RNA and proteins // United States Patent 5945515 (1999).

18. Chetverina H.V., Samatov T.R., Ugarov V.I., Chetverin A.B. Molecular colony diagnostics: Detection and quantitation of viral nucleic acids by in-gel PCR // BioTechniques. V.33. P.150-156 (2002).

19. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction // Anal. Biochem. V.162. P.156-159 (1987).

20. Boom R., Sol C. J., Salimans M. M., Jarisen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids // J. Clin. Microbiol. V.28. P. 495-503 (1990).

Claims (13)

1. A method of preserving biological samples, comprising obtaining a lysate by mixing the biological material with a preservation solution containing a guanidine compound in an amount sufficient to substantially completely denature the nucleases contained in the sample, characterized in that the preservation solution also contains the following components that prevent the degradation of nucleic acids: (1) a reducing agent for protein SS bonds in an amount providing substantially complete restoration of SS bonds in lysate proteins; (2) a strong detergent that does not form an insoluble compound with guanidine in an amount sufficient to form micelles in the lysate; (3) at least one complex of ions of divalent and trivalent metals in an amount providing its molar concentration in the lysate is obviously higher than the expected maximum molar concentration of ions of divalent and trivalent metals; (4) a buffer that maintains the pH of the lysate in the range of 6 to 8. 2. The method according to claim 1, characterized in that during mixing with a preservative solution, the biological material is homogenized. 3. The method according to claim 1, characterized in that the substance selected from the group of substances including 2-mercaptoethanol, dithiothreitol, thiourea and glutathione is used as a reducing agent for protein S-S bonds. 4. The method according to claim 1, characterized in that 2-mercaptoethanol is used as a reducing agent for protein S-S bonds. 5. The method according to claim 1, characterized in that as a detergent use a substance selected from the group of substances including sarcosyl, polyethylene glycol ethers and polyoxyethylene sorbitans. 6. The method according to claim 1, characterized in that sarcosyl is used as a detergent. 7. The method according to claim 1, characterized in that the complexon is a substance selected from the group of substances including EDTA, EGTA, 8-hydroxyquinoline and 1,10-phenanthroline. 8. The method according to claim 1, characterized in that EDTA is used as complexon. 9. The method according to claim 1, characterized in that a citric acid salt is used as a buffer. 10. The method according to claim 1, characterized in that the resulting lysate is mixed with a water-soluble organic solvent capable of causing precipitation of nucleic acids, taken in an amount sufficient to ensure substantially complete precipitation of the nucleic acids present in the lysate. 11. The method according to claim 10, characterized in that as an organic solvent use a substance selected from the group of substances including low molecular weight alcohols, acetone, polyethylene glycol and dimethyl sulfoxide. 12. The method according to claim 10, characterized in that isopropanol is used as the organic solvent. 13. The method according to p. 12, characterized in that the volume of added isopropanol is twice the volume of the lysate.

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