Abstract
RNA is a tool used in many fields, from molecular and cellular biology to medicine and nanotechnology. For most of these uses, the integrity of RNA is required and must be maintained during storage. Even though freezing is currently the storage method of choice, the increasing number of samples to be stored and the costly use of a cold chain have highlighted the need for room temperature preservation methods. Here, we report a new room temperature technology that consists in drying RNA samples in the presence of a stabilizer in stainless steel minicapsules. These air- and water-tight capsules isolate RNA from the atmosphere and maintain an anhydrous and anoxic environment. Through the evaluation of RNA integrity over time at room temperature or 90 °C, we identified atmospheric humidity as a major deleterious factor. The degradation rate dependence in temperature fitted an Arrhenius model, with an activation energy of 28.5 kcal/mol and an extrapolated room temperature degradation rate of 3.2 10−13/nt/s (95% confidence interval: 2.3–4.2/nt/s). In these conditions, it is expected that an RNA molecule will be subjected to 0.7–1.3 cut every 1000 nucleotides per century. In addition, we showed that stored RNA is compatible for further analyses, such as reverse transcription-quantitative PCR. No significant change in the Cq values was observed over a simulated period of several decades. At last, our data are consistent with a sequence-independent degradation rate of RNA in the solid state.
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References
Madej RM, Davis J, Holden MJ, Kwang S, Labourier E, Schneider GJ : International standards and reference materials for quantitative molecular infectious disease testing. J Mol Diagn 2010; 12: 133–143.
White HE, Matejtschuk P, Rigsby P et al: Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA. Blood 2010; 116: e111–e117.
Baginsky S, Hennig L, Zimmermann P, Gruissem W : Gene expression analysis, proteomics, and network discovery. Plant Physiol 2010; 152: 402–410.
Malone J, Oliver B : Microarrays, deep sequencing and the true measure of the transcriptome. BMC Biol 2011; 9: 34.
Wang Z, Gerstein M, Snyder M : RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 2009; 10: 57–63.
Poulsen HE, Specht E, Broedbaek K et al: RNA modifications by oxidation: a novel disease mechanism? Free Radic Biol Med 2012; 52: 1353–1361.
Bruskov VI, Malakhova LV, Masalimov ZK, Chernikov AV : Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA. Nucleic Acids Res 2002; 30: 1354–1363.
Evans RK, Xu Z, Bohannon KE, Wang B, Bruner MW, Volkin DB : Evaluation of degradation pathways for plasmid DNA in pharmaceutical formulations via accelerated stability studies. J Pharm Sci 2000; 89: 76–87.
Cataldo F : Ozone degradation of ribonucleic acid (RNA). Polym Degrad Stabil 2005; 89: 274–281.
Emilsson GM, Nakamura S, Roth A, Breaker RR : Ribozyme speed limits. RNA 2003; 9: 907–918.
Oivanen M, Kuusela S, Lonnberg H : Kinetics and mechanisms for the cleavage and isomerization of the phosphodiester bonds of RNA by bronsted acids and bases. Chem Rev 1998; 98: 961–990.
Perreault DM, Anslyn EV : Unifying the current data on the mechanism of cleavage–transesterification of RNA. Angew Chem Int Ed 1997; 36: 432–450.
Seyhan AA, Burke JM : Mg2+-independent hairpin ribozyme catalysis in hydrated RNA films. RNA 2000; 6: 189–198.
Soukup GA, Breaker RR : Relationship between internucleotide linkage geometry and the stability of RNA. RNA 1999; 5: 1308–1325.
Kaukinen U, Lyytikainen S, Mikkola S, Lonnberg H : The reactivity of phosphodiester bonds within linear single-stranded oligoribonucleotides is strongly dependent on the base sequence. Nucl Acids Res 2002; 30: 468–474.
Ma S, Huang Y, van Huystee RB : Improved plant RNA stability in storage. Anal Biochem 2004; 326: 122–124.
Kazakov SA, Balatskaya SV, Johnston BH : Ligation of the hairpin ribozyme in cis induced by freezing and dehydration. RNA 2006; 12: 446–456.
Baker M : Biorepositories: building better biobanks. Nature 2012; 486: 141–146.
Wan E, Akana M, Pons J et al: Green technologies for room temperature nucleic acid storage. Curr Issues Mol Biol 2009; 12: 135–142.
Natarajan P, Trinh T, Mertz L, Goldsborough M, Fox DK : Paper-based archiving of mammalian and plant samples for RNA analysis. Biotechniques 2000; 29: 1328–1333.
Novoradovskaya N, Basehore L, Scott Braman JC Stabilization of nucleic acids on solid supports 2009, WO 2009/029433 A2.
Bates M, Goldsborough A RNA sample stabilization in the presence of a transitional metal 2011, WO 2011/008553 A1.
Bonnet J, Colotte M, Coudy D et al: Chain and conformation stability of solid-state DNA: implications for room temperature storage. Nucleic Acids Res 2010; 38: 1531–1546.
Colotte M, Coudy D, Tuffet S, Bonnet J : Adverse effect of air exposure on the stability of dna stored at room temperature. Biopreserv Biobank 2011; 9: 47–50.
Fleige S, Pfaffl M : RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med 2006; 27: 126–139.
Byrn SR, Xu W, Newman AW : Chemical reactivity in solid-state pharmaceuticals: formulation implications. Adv Drug Deliv Rev 2001; 48: 115–136.
Shalaev EY, Zografi G : How does residual water affect the solid-state degradation of drugs in the amorphous state? J Pharm Sci 1996; 85: 1137–1141.
Bhattacharya S, Suryanarayanan R : Local mobility in amorphous pharmaceuticals—characterization and implications on stability. J Pharm Sci 2009; 98: 2935–2953.
Falk M : The ultraviolet spectra of ribonucleic acids in solid state and in solution. Rev Canadienne Chim 1965; 43: 3151–3159.
Brisco MJ, Morley AA : Quantification of RNA integrity and its use for measurement of transcript number. Nucleic Acids Res 2012; 40: e144.
Li Y, Breaker RR : Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2'-hydroxyl group. J Am Chem Soc 1999; 121: 5364–5372.
Imbeaud S, Graudens E, Boulanger V et al: Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucl Acids Res 2005; 33: e56.
Fleige S, Walf V, Huch S, Prgomet C, Sehm J, Pfaffl MW : Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol Lett 2006; 28: 1601–1613.
Vermeulen J, De Preter K, Lefever S et al: Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res 2011; 39: e63.
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B : Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Meth 2008; 5: 621–628.
Das M, Harvey I, Chu LL, Sinha M, Pelletier J : Full-length cDNAs: more than just reaching the ends. Physiol Genomics 2001; 6: 57–80.
Acknowledgements
We thank D Coudy for help and discussions, the ENSTBB, Marc Bonneu for discussions and making available a qPCR machine, Pr. Merlio and Y Drissi for supplying oligonucleotides and Integragen for sharing their Bioanalyzer. This work was supported by the ‘Conseil Régional d’Aquitaine’, ‘FEDER’, ‘Genopole’ and ‘Oséo Innovation’
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S Tuffet is CEO and shareholder of Imagene Company; M Colotte, A-L Fabre, A Luis are employees of Imagene Company; J Bonnet, is a shareholder, has consulted for Imagene Company and received compensation.
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Fabre, AL., Colotte, M., Luis, A. et al. An efficient method for long-term room temperature storage of RNA. Eur J Hum Genet 22, 379–385 (2014). https://doi.org/10.1038/ejhg.2013.145
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DOI: https://doi.org/10.1038/ejhg.2013.145
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