The precision and reliability of quantitative nucleic acid analysis depends on the quality of the sample analyzed and the integrity of the nucleic acids. The integrity of RNA is currently primarily assessed by the analysis of ribosomal RNA, which is the by far dominant species. The extrapolation of these results to mRNAs and microRNAs, which are structurally quite different, is questionable. Here we show that ribosomal and some nucleolar and mitochondrial RNAs, are highly resistant to naturally occurring post-mortem degradation, while mRNAs, although showing substantial internal variability, are generally much more prone to nucleolytic degradation. In contrast, all types of RNA show the same sensitivity to heat. Using qPCR assays targeting different regions of mRNA molecules, we find no support for 5' or 3' preferentiality upon post-mortem degradation.

Laboratory of Gene Expression Institute of Biotechnology Academy of Sciences of the Czech Republic Videnska 1083 142 20 Prague Czech Republic

Laboratory of Gene Expression Institute of Biotechnology Academy of Sciences of the Czech Republic Videnska 1083 142 20 Prague Czech Republic; Charles University Prague Faculty of Science Department of Cell Biology Vinicna 7 128 43 Prague Czech Republic

Laboratory of Gene Expression Institute of Biotechnology Academy of Sciences of the Czech Republic Videnska 1083 142 20 Prague Czech Republic; TATAA Biocenter AB Odinsgatan 28 411 03 Göteborg Sweden

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Sampaio-Silva F., Magalhaes T., Carvalho F., Dinis-Oliveira R.J., Silvestre R. Profiling of RNA degradation for estimation of post mortem interval. PLoS One. 2013;8:e56507. PubMed PMC

Malentacchi F., Pazzagli M., Simi L., Orlando C., Wyrich R., Hartmann C.C., Verderio P., Pizzamiglio S., Ciniselli C.M., Tichopad A., Kubista M., Gelmini S. SPIDIA-DNA: an external quality assessment for the pre-analytical phase of blood samples used for DNA-based analyses. Clin. Chim. Acta. 2013;424:274–286. PubMed

Malentacchi F., Pazzagli M., Simi L., Orlando C., Wyrich R., Günther K., Verderio P., Pizzamiglio S., Ciniselli C.H.M., Zhang H., Korenkova V., Rainen L., Bar T., Kubista M., Gelmini S. Second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses. PLoS One. 2014;9(11):e112293. PubMed PMC

Zhang H., Korenkova V., Sjöback R., Svec D., Björkman J., Kruhøffer M., Verderio P., Pizzamiglio S., Ciniselli C.H.M., Wyrich R., Oelmueller U., Kubista M., Lindahl T., Lönneborg A., Rian E. Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples. PLoS One. 2014;9(11):e111644. PubMed PMC

Raeymarkers L. Quantitative PCR: theoretical consideration with practical implication. Anal. Biochem. 1993;214:582–585. PubMed

Imbeaud S., Graudens E., Boulanger V., Barlet X., Zaborski P., Eveno E., Mueller O., Schroeder A., Auffray C. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 2005;33:e56. PubMed PMC

Fleige S., Pfaffl M.W. RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Aspects Med. 2006;27:126–139. PubMed

Fleige S., Walf V., Hunch S., Prgomet C., Sehm J., Pfaffl M.W. Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol. Lett. 2006;28:1601–1613. PubMed

Strand C., Enell J., Hedenfalk I., Ferno M. RNA quality in frozen breast cancer samples and the influence on gene expression analysis—a comparison of three evaluation methods using microcapillary electrophoresis traces. BMC Mol. Biol. 2007;8:38. PubMed PMC

Takano E.A., Mikeska T., Dobrovic A., Byrne D.J., Fox S.B. A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues. BMC Biotechnol. 2010;10:89. 89. PubMed PMC

Gallego Romero I., Pai A.A., Tung J., Gilad Y. RNA-seq: impact of RNA degradation on transcript quantification. BMC Biol. 2014;12:42. PubMed PMC

Larson D.E., Zahradka P., Sells B.H. Control points in eukaryotic ribosome biogenesis. Biochem. Cell Biol. 1991;69:5–22. PubMed

Sambrook J., Fritsch E.F., Maniatis T. 2nd edn. Cold Spring Harbor Laboratory Press; NY: 1989. Molecular Cloning: A Laboratory Manual.

Mueller O., Lightfoot S., Schroeder A. RNA integrity number (RIN)—standardization of RNA quality control application. Agilent Application Note. 2004:1–8. Publication 5989-1165EN.

Miller C.L., Diglisic S., Leister F., Webster M., Zolken R.H. Evaluating RNA status for RT-PCR in extracts of postmortem human brain tissue. Biotechniques. 2004;36:628–633. PubMed

Mori N., Mizuno D., Goto S. Increase in the ratio of 18S RNA to 28S RNA in the cytoplasm of mouse tissues during aging. Mech. Ageing Dev. 1978;8:285–297. PubMed

Houge G., Robaye B., Eikhom T.S., Golstein J., Mellgren G., Gjertsen B.T., Lanotte M., Doskeland S.O. Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis. Mol. Cell Biol. 1995;15:2051–2062. PubMed PMC

Schroeder A., Mueller O., Stocker S., Salowsky R., Leiber M., Gassmann M., Lightfoot S., Menzel W., Granzow M., Ragg T. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 2006;7:3. PubMed PMC

Bauer M. RNA in forensic science. Forensic Sci. Int. Genet. 2007;1:69–74. PubMed

Ibberson D., Benes V., Muckenthaler M., Castoldi M. RNA degradation compromises the reliability of microRNA expression profiling. BMC Biotechnol. 2009;9:102. PubMed PMC

Fordyce S.L., Kampmann M.L., van Doorn N.L., Gilbert M.T. Long-term RNA persistence in postmortem contexts. Investig. Genet. 2013;4:7. PubMed PMC

Yamashita A., Chang T.C., Yamashita Y., Zhu W., Zhong Z., Chen C.Y., Shyu A.B. Concerted action of poly(A) nuclaeses and decapping enzymes in mammalian mRNA turnover. Nat. Struct. Mol. Biol. 2005;12:1054–1063. PubMed

Schoenberg D.R. Mechanisms of endonuclease-mediated mRNA decay. Wiley Interdiscip. Rev. RNA. 2011;2:582–600. PubMed PMC

Garneau N.L., Wilusz J., Wilusz C.J. The highways and byways of mRNA decay. Nat. Rev. Mol. Cell Biol. 2007;8(2):113–126. PubMed

Beelman C.A., Parker R. Degradation of mRNA in eukaryotes. Cell. 1995;81(2):179–183. PubMed

Lubas M., Damgaard C.K., Tomecki R., Cysewski D., Jensen T.H., Dziembowski A. Exonuclease hDIS3L2 specifies an exosome-independent 3′–5′ degradation pathway of human cytoplasmic mRNA. EMBO J. 2013;32(13):1855–1868. PubMed PMC

Matera A.G., Terns R.M., Terns M.P. Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs. Nat Rev Mol Cell Biol. 2007;8(3):209–220. PubMed

Differential amplicons (ΔAmp)-a new molecular method to assess RNA integrity