Highly degraded RNA can still provide molecular information: An in vitro approach

Abstract

The long‐term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR‐based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical–chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat‐mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di‐ethyl‐pyro‐carbonate‐water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60 bp long) housekeeping targets (glyceraldehyde‐3‐phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r2 > 0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end‐point PCR of blood‐specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR‐based results show that RNA maintains the ability to be retro‐transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long‐term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed.

Assessment of RNA extraction protocols from cladocerans

“…Furthermore, the occurrence of RNases in both the sample and the environment causes the breakdown of RNA into smaller components [ 56 ]. The hydroxyl (-OH) group and diatomic carbon (C2) group in RNA chemical structure play a crucial role in the nonenzymatic degradation process [ 71 ]. Degraded RNA will not become amplified at the same level as cDNA derived from intact RNA during gene expression analysis.…”

The production and characterization of SARS-CoV-2 RNA reference material

“…The in vitro transcribed SARS-CoV-2 synthetic RNA was diluted in 1 mM sodium citrate buffer (pH: 6.5, diethylpyrocarbonate treated and autoclaved) containing 2.5 ng/μL total human control RNA (Thermo Fisher 4307281) to an approximate concentration of 10 8 copies/μL (according to the Qubit RNA BR assay kit (Quant-iT™) measurement) and the tubes were filled in 100 μL volume [22]. All mixing and filling operations were carried out on ice.…”

Nucleic Acids Persistence—Benefits and Limitations in Forensic Genetics

“…RNA is hydrolyzed in both acidic and alkaline conditions, while DNA is only hydrolyzed in acidic conditions [ 8 , 9 ]. After death, RNA is degraded by ribonuclease naturally occurring in the cell [ 10 ] or originating from bacteria and other environmental contaminants. The physical and non-specific chemical factors exacerbate this effect.…”