Effects of post-mortem and physical degradation on RNA integrity and quality

Abstract

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.

Keywords: Degradation; RNA integrity; RNA quality; RQI; RT-qPCR.

Fig. 1

Experion electropherograms of total RNAs and determined RQI values for post-mortem degraded (A.) oocytes and (B.) tadpole embryos at stage 40 measured at intervals 0, 5, 10, 20 and 40 min post-mortem.

Fig. 2

Temporal degradation profiles of unstable genes (in blue – odc, imp3, RNA pol. II, maml1, atub, acta, eef1a1, mrpl1, ubc, ant1, mdh2a, xk81a1) – shown in (A.) oocyte and (B.) tadpole at stage 40. Degradation profiles of stable genes (in orange – scaRNA11, 5S rRNA, cyc1 and 18S rRNA) – (C.) oocyte and (D.) tadpole at stage 40. Axes x in all graphs indicate intervals of post-mortem samples in minutes and axes y indicate relative quantity transferred to log scale. Profiles for stable genes were averaged in panels (A. and B.), and profiles for unstable genes were averaged in panels (C. and D.) to simplify comparison (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 3

Statistical analysis of degradation profiles. (A.) SOM analysis and (B.) hierarchical clustering for stable (orange) and degraded (blue) transcripts in post-mortem samples. RNA spike, which was added before reverse transcription, is included in analysis to indicate stable RNA level (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 4

Five different qPCR assays covering the whole molecules of (A.) xk81a1 and (B.) atub were designed and quantified to determine 5′ and 3′ specific degradation. Axis x indicates post-mortem intervals in minutes and axis y indicates relative quantity.

Fig. 5

Heat degradation. Extracted RNAs from embryos were heated for 0, 1, 2, 4 and 6 h (axis x) at 80 °C. (A.) Total RNA quality and RQI was tested by Experion system. (B.) Temporal degradation profiles of 16 transcripts measured using RT-qPCR. Post-mortem unstable genes are shown in blue (odc, imp3, RNA pol. II, maml1, atub, acta, eef1a1, mrpl1, ubc, ant1, mdh2a, xk81a1) and stable genes are shown in orange (scaRNA11, 5S rRNA, cyc1 and 18S rRNA).