WO2012075471A1

WO2012075471A1 - Rna stability enhancer

Info

Publication number: WO2012075471A1
Application number: PCT/US2011/063189
Authority: WO
Inventors: Hui-Yu Liu
Original assignee: Hui-Yu Liu
Priority date: 2010-12-04
Filing date: 2011-12-02
Publication date: 2012-06-07
Also published as: CN103370412A; CN103370412B

Abstract

This disclosure relates to reagents, compositions, methods for further stabilizing RNA in sample lysates by supplementing lysis buffer with RNase inhibitory compound(s).

Description

RNA STABILITY ENHANCER

Field of the invention

[001] The present invention relates to the methods, compositions and kits for enhanced and prolonged stability of nucleic acids, in particular RNA, in lysates and for the integrated workflow from sample collection, homogenization, storage, transportation and to the final nucleic acids extraction.

Background of the invention

[002] The preservation of the integrity and profile of biomolecules during biomolecules isolation from biological samples is critical for the success of biological and clinical investigations. This however is challenged by the fact that biomolecules are designed to undergo dynamic and highly regulated metabolism. Cells and tissues utilize compartment of and controlled release of degrading agents to regulate the turnover of biomolecules. The enzymatic processes are responsible for the majority of the biomolecules degradations. The abundant and robust ribonucleases (RNases) are the main reason for RNAs to be extremely liable to degradations. RNases are a large group of ubiquitous enzymes, certain tissues like the pancreas are particularly abundant in RNases. RNase are very stable even surviving the boiling for 20 min (Berkower, (1973) Isolation and Characterization of an Endonuclease from Escherichia coli Specific for Ribonucleic Acid in Ribonucleic Acid'Deoxyribonucleic Acid Hybrid Structures. J. Biol. Chem. 248(17): 5914-5921.) In the process of isolation of biomolecules, cell compartments are disrupted in order to release the biomolecules, this however exposes the interested biomolecules to their respective digestive agents, like RNA to RNases, leading to the quick degradation of the biomolecules. To overcome this obstacle, most RNA extraction methods adopt extreme harsh chemicals to deactivate RNases from biological samples during the lysis stage, chemicals including phenol and high concentration chaotropic salts, like 4~5 of guanidine are frequently used in lysis buffer to deactivate RNases (Katayanagi K et al., (1990) Three-dimensional structure of ribonuclease H from E. coli. Nature 347(6290): 306-309). Other strong denaturants like IiCl and SDS are also used in the lysis buffers.

[003] Even with above mentioned strong RNase denaturants (e.g. 4~5M guanidine) RNA still undergo progressive and significant degradation in lysates, this makes it necessary to process the RNA isolation immediately after the sample homogenization or keep the lysates at low temperature.

[004] Because of the continuous RNA degradation, the guanidine containing lysis solutions are thus only used for tissue / cell lysis that is immediately followed by nucleic acids isolation, they are rarely used for the biological sample storage, archiving or transportation purpose, instead, high concentration of salts, like ammonium sulphate in RNAlater™ or detergents in PAXgene blood tubes, are developed to store, archive and transport samples with RNA as interested contents.

[005] The issue with most currently available RNA containing sample storage solutions is that they are not compatible with common downstream RNA isolation methods: RNAlater has to be completely removed from samples using tissue towels; and RNA containing pellets need to be precipitated from PAXgene tubes before lysis solutions being added to isolate RNA from samples. [006] It is obviously desired to enhance and prolong the RNA stability in common lysis solutions doe the purpose of the integrated sample collection, storage, archiving, transportation and the final nucleic acids isolation.

[007] It is widely assumed that no enzyme activities would remain within 4~5M guanidine solutions, so the RNA degradation with it would rather be caused by spontaneous, alkali or divalent metal cations mediated hydrolysis, or by some secondary products of guanidine and biological material reactions. We however suspected mat mere are residual RNase activities that can be further inhibited. It is supported by the observations that RNases are still active in 8M urea or 6 guanidine hydrochloride (Darlix, J. L. (1975) Simultaneous Purification of Escherichia coli Termination Factor Rho, RNAase III and RNAase H. Eur. J. Biochem. 51(2), 369-376.; Barone G et al., (1994) RNAase A in the presence of urea and GuHCl. J. Thermal Analysis. 41: 1357-1370.). This prompted us to look for RNase inhibitors that can remain functional in lysis buffers and provide further RNase inhibition.

[008] Some of the RNase inhibitory compounds reported in literatures are: Pyrophosphate compounds (Russo, N., et al., (19%) A combined kinetic and modeling study of the catalytic center subsites of human angiogenin. Proc. Natl. Acad. Sci. 93: 804-808; Russo A et a., (2001) Small molecule inhibitors of RNase A and related enzymes. Methods Enzymol. 341: 629-648; Leonidas et. al., (2001) Binding of Phosphate and pyrophosphate ions at the active site of human angiogenin as revealed by X-ray crystallography. Protein Science 10(8): 1669-1676.); and copper ion (Nishimura et. al., (1994) Utilization of copper ion as a ribonuclease inhibitor in a cell-free protein synthesis system. Journal of Fermentation and Bioengineering 78(2): 130-133); and organic compounds like C- 181431 from ChemBridge ( ao et. al., A small-molecule inhibitor of the ribonucleolytic activity of human angiogenin that possesses antitumor activity (2002) Proc Natl Acad Sci U S A. 99(15): 10066-10071).

[009] The present invention provides the desired nucleic acids stability enhancement by adding one or more RNase inhibitory compounds to common lysis solutions.

Brief Description of the Drawings

[0010] Figure 1. 1.2% EtBr Agarose gel analysis of RNA extracted using Qiagen RNeasy mini kit from mouse liver lysates that were incubated at 37 °C for the indicated durations. Results demonstrated the improved RNA stability with addition of lOmM each of various RNase inhibitory compounds in the RLT lysis buffer.

[0011] Figure 2. A. Agarose gel electrophoresis and B. Agilent Nano chip RIN index of RNA using GE Illustra RNAspin Mini RNA Isolation kit extracted from mouse liver lysates that were incubated at 37 °C for the indicated durations. Results demonstrated the improved RNA stability with the presence of 10 inM NaPPi in lysis buffer.

[0012] Figure 3. Agilent Pico chip gel image of RNA extracted from HeLa cells that were spotted on Whatman® paper and incubated at 37 °C for 4 hours, the Whatman® paper patches were soaked with lysis buffer from GE Illustra RNA Isolation kit and various concentration of sodium pyrophosphate, the filter paper patches were dried before HeLa cell suspension were dropped onto. Results demonstrated the improved RNA stability on filter paper that was soaked with lysis buffer with 10 mM and higher NaPPi. Summary of the Invention

[0013] This disclosure relates, in part, to reagents, compositions, and /or methods for stabilizing RNA in biological samples lysates by supplementing one or more (e.g., combination of two, three, four, etc.) RNase inhibitory compound(s) to common tissue/cell lysis buffers that contain high concentration of chaotropic agent (e.g., guanidine). In some aspects, the additive compound(s) is/are premixed with lysis buffer and stored at room temperature for at least one week, in other aspects, the additive compound(s) is/are added in lysis buffer right before the homogenization of tissues/cells, in still other aspects, the additive compound(s) is/are added right after the tissues/cells homogenization in common lysis buffers. In some aspects, the supplemented compound is sodium pyrophosphate, in the other aspects, the supplemented compound is copper chloride, in still other aspects, the supplemented compound is 4,4'- carbonylbis[2-(l-naphthoylamino)benzoic acid].

Detailed Description of the Invention

[0014] In one aspect, the reagents, compositions and methods comprise and/or involve the addition of RNase inhibitory compound(s). The selection criteria for appropriate RNase inhibitory compounds are: enhance RNA stability in lysates; maintain or even boost RNA yields regardless of the downstream extraction methods (e.g., glass filters or magnetic beads, etc.); do not render preference for the recovery of one subpopulation of RNA over the others; do not cause interference to the downstream applications (e.g., RT-PCR, Northern blot, etc.); do not cause the modification of RNA (e.g., depurination etc.); do not increase the co-purification of impurities (e.g. DNA, proteins, lipids, etc.); soluble in lysis buffer to achieve functional concentration (e.g., 20 mM, 15 mM, 10 mM or lower) and stable in lysis buffer; do not cause precipitation in lysis buffer immediately or after period of storage; non-toxic; relatively cheap; easy to be washed off during the following wash steps.

[0015] It will be evident that new and more RNase inhibitory compounds can be identified or made. It will be not difficult for one who is skilled in the field to screen for the RNase inhibitory compounds. The inhibition of RNase can be easily observed by, for example, using RNaseAlert assay (Ambion), in which an RNA substrate is dual labeled with fluorescence and quencher on both ends, increased fluorescence can be observed using fluorescence plate reader when the RNA substrate are degraded by co-existent RNase(s), the functional RNase inhibitors) will decrease the result fluorescence when co-presented with RNase(s) and RNA substrate. The screening can also be done by running the recovered RNA in agarose gel or RNA chips on Agilent Bioanalyzer2100 for visualization of the integrity of RNA, or by Ct values from q-QT- PCR.

[0016] It will be evident that known or new RNase inhibitory compounds other than those included in this disclosure can be added into lysis buffer and enhance RNA stability. It will be easy for one who is skilled in the art to test different RNase inhibitory compounds can be tested for their suitability in this invention by adding them into lysis buffer and analyze the integrity and yield of resulted RNA. The enhanced RNA stability in lysates with addition of extra RNase inhibitory compound(s) could be tested and evidenced by homogenizing tissue (e.g., mouse liver, pancreas, etc.) in common lysis buffer with chaotropic agent (e.g. RLT from Qiagne's RNeasy RNA kit), tissue lysates with or without addition of extra RNase inhibitory compound(s) are then incubated at, for example, 65 °C for several hours, or 37 °C for a day or longer, or room temperature for a day or longer, the RNA from the lysates are then extracted according to the RNA kit protocol. The integrity of the recovered RNA is then visualized and quantitated by agarose gel electrophoresis or by running RNA chips on a Bioanalyser2100 (Agilent). The RNA integrity can also be analyzed by running quantitative RT-PCR and judged by Ct values. The yield of RNA can be determined by OD260 spectrometry or Bioanalyser2100.

Examples

RNA stabilization in mouse liver lysates with addition of pyrophosphate in RLT lysis buffer of Qiagen RNeasy mini kit.

[0017] 20 milligrams of previously -80 °C preserved mouse liver were homogenized using polytron in 350μ1 of Buffer RLT (QIAGEN RNeasy Mini Kit, Cat. No. 74106) containing 1% of 14.3M β-mercaptoet anol, with or without the addition of 7 ul 0.5 M sodium pyrophosphate dibasic (Sigma-Aldrich Cat. No. P8135), the addition of pyrophosphate solution could be long before liver homogenization and kept at room temperature for at least one week, or right before liver homogenization, or right after the homogenization. The lysates were incubated at 37 °C for the indicated duration and kept in -20 °C until the last time point was reached. All aliquots were then thawed and processed for RNA according to manufacturer's instructions. The recovered RNA were equal volume mixed with glyoxal gel loading buffer (Ambion, Cat. No. AM8551), and incubated at 50 °C for 30 min before loaded onto 1.2% agarose gel for electrophoresis (Fig. 1). RNA stabilization in mouse liver lysates with addition of copper ion in RLT lysis buffer of Qiagen RNeasy mini kit.

[0018] 20 milligrams of previously -80 °C preserved mouse liver were homogenized using polytron in 350ul of Buffer RLT (QIAGEN RNeasy Mini Kit, Cat No. 74106) containing 1% of 14.3M β-mercaptoethanol, with or without the addition of 7 ul 0.5 M copper chloride (Sigma- Aldrich Cat. No. 203149), the addition of copper chloride solution could be long before liver homogenization and kept at room temperature for at least one week, or right before liver homogenization, or right after the homogenization. The lysates were incubated at 37 °C for the indicated duration and kept in -20 °C until the last time point was reached. All aliquots were then thawed and processed for RNA according to manufacturer's instructions. The recovered RNA were equal volume mixed with glyoxal gel loading buffer (Ambion, Cat No. AM8551), and incubated at 50 °C for 30 min before loaded onto 12Vo agarose gel for electrophoresis (Fig. 1).

RNA stabilization in mouse liver lysates with addition of 4,4'-carbonylbis[2-(l- naphmoylamino)benzoic add] in RLT lysis butter of Qiagen RNeasy mini kit.

[0019] 20 milligrams of previously -80 °C preserved mouse liver were homogenized using polytron in 350ul of Buffer RLT (QIAGEN RNeasy Mini Kit, Cat No. 74106) containing 1% of 14.3M β-mercaptoethanol, with or without the addition of 7 ul 0.5 M 4,4'-carbonylbis[2-(l- naphthoylamino)benzoic acid] in DMSO (ChemBridge Cat. No. 5181431), the addition of the compound solution could be long before liver homogenization and kept at room temperature for at least a week, or right before liver homogenization, or right after the homogenization. The lysates were incubated at 37 °C for the indicated duration and kept in -20 °C until the last time point was reached. All aliquots were then thawed and processed for RNA according to manufacturer's instructions. The recovered RNA were equal volume mixed with glyoxal gel loading buffer (Ambion, Cat. No. AM8551), and incubated at 50 °C for 30 min before loaded onto 1.2% agarose gel for electrophoresis (Fig. 1).

RNA stabilization in mouse liver lysates with addition of pyrophosphate in lysis buffer of GE Dlustra RNAspin Mini RNA Isolation kit.

[0020] 20 milligrams of previously -80 °C preserved mouse liver were homogenized using polytron in 350ul of Buffer RA1 (GE Healthcare, #25-0500-71) containing 1% of 14.3M β- mercaptoethanol, with or without the addition of 7 ul 0.5 M sodium pyrophosphate dibasic (Sigma-Aldrich Cat. No. P8135), the addition of pyrophosphate solution could be long before liver homogenization and kept at room temperature for at least one week, or right before liver homogenization, or right after the homogenization. The lysates were incubated at 37 °C for the indicated duration and kept in -20 °C until the last time point was reached. All aliquots were then thawed and processed for RNA according to manufacturer's instructions. The recovered RNA were equal volume mixed with glyoxal gel loading buffer (Ambion, Cat No. AM8551), and incubated at 50 °C for 30 min before loaded onto 1.2% agarose gel for electrophoresis(Fig. 2A). The recovered RNA were also denatured at 70 °C for 2min and ran on Agilent Nano RNA chips (Agilent, #5067-1512) according to manufacturer's instructions, the RIN index indicate the RNA integrity (Fig. 2B).

RNA stabilization in cell lysates on dry solid matrix that were soaked with RNase inhibitory compounds enhanced lysis buffer.

[0021] Common Whatman® filter paper were soaked with water, or lysis Buffer RA1 (GE Healthcare, #25-0500-71) containing 0, 5, 10, 20, 25, 50, or 100 niM of sodium pyrophosphate dibasic (NaPPi, Sigma-Aldrich Cat No. P8135) as additional RNase inhibitory compound, the soaked filter paper were lift out of the solution and dried at room temperature overnight. 5 X 10⁵ / uL HeLa cells in PBS were dropped onto dried filter paper and the FTA® card (Whatman, WB120310). HeLa cells on paper were incubated at 37 °C for 4 hours. Three punches were taken from each filter paper patch, and HeLa RNA were recovered using Illustra® RNAspin Mini RNA Isolation kit (GE Healthcare, #25-0500-71) according to manufacturer's instructions. The recovered RNA were denatured at 70 °C for 2min and ran on Agilent Pico RNA chip (Agilent, #5067-1514) according to manufacturer's instructions, the RTN index indicate the RNA integrity (Fig. 3).

[0022] It would be apparent to one skilled in the art that the liver sample can be replaced with other tissues and cell types as recommended by the kit instructions. With the enhanced RNA stability, some other sample types might become appropriate to be added to the recommended sample types.

[0023] It will be apparent to one skilled in the art that the type and concentration of the RNase inhibitory compounds are not limited to those listed in above examples.

[0024] It will be apparent to one skilled in the art that the type of solid supporting matrix for RNase inhibitory compounds enhanced lysis buffer can be all kinds of suitable materials that are not limited to Whatman® filter paper that was used in above example.

Claims

1. A method for further stabilizing RNA in the lysates of RNA-containing samples by supplementing lysis buffer with RNase inhibitor(s).

2. A method according to claim 1, wherein the sample lysis buffers contain high concentration of chaotropic salts.

3. A method according to claim 1, wherein the concentration of chaotropic salts is more than 2 M and less than 8 M.

4. A method according to claim 2, wherein the chaotropic salts are guanidine thiocyanate.

5. A method according to claim 2, wherein the chaotropic salts are guanidine hydrochloride.

6. A method according to claim 1, wherein the RNase inhibitors are derived from known or newly identified RNase inhibitors.

7. A method according to claim 1, wherein the RNase inhibitors are pyrophosphate compounds.

8. A method according to claim 1, wherein the RNase inhibitors are copper compounds.

9. A method according to claim 1, wherein the RNase inhibitor is 4,4'-carbonylbis[2-(l- naphthoylamino)benzoic acid].

10. A method according to claim 1, wherein the RNase inhibitors) supplemented lysis buffer is dried onto solid supporting matrix.

11. A method according to claim 1, wherein the RNA are mRNA, tRNA, rRNA, non -coding RNA, small RNA, si RNA, miRNA, animal RNA, plant RNA, viral RNA, bacterial RNA.

12. A kit for extracting RNA from a biological sample, which kit comprises guanidine and RNase inhibitor(s) in its sample lysis buffer, wherein the RNase inhibitor(s) is/are derived from known or newly identified RNase inhibitors.

13. A kit according to claim 12, wherein the RNase inhibitors are pyrophosphate compounds, and / or copper compounds, and / or 4, 4'-carbonylbis [2-(l-naphthoylamino) benzoic acid].

14. A kit according to claim 12, wherein the RNase inhibitors) are premixed in guanidine containing lysis buffer.

15. A kit according to claim 12, wherein the RNase inhibitor(s) is/are provided as a separate concentrated solution(s) for addition to the guanidine containing lysis solution.

16. Use of a combination of guanidine and RNase inhibitors) for further stabilizing RNA in the lysates of RNA containing biological samples.