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  • Highly degraded RNA can still provide molecular information : an in vitro approach
    Fattorini, Paolo ...
    The long-term survival of RNA in post mortem 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 hours using ultrapure water and di-ethyl-pyro-carbonate (DEPC)-water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The RT/qPCR 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 hours 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, acidity, etc.) 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.
    Vir: Electrophoresis. - ISSN 0173-0835 (no. 516, Vol. 41, Apr. 2020, str. 386-393)
    Vrsta gradiva - članek, sestavni del ; neleposlovje za odrasle
    Leto - 2020
    Jezik - angleški
    COBISS.SI-ID - 34662873

    Povezava(-e):

    https://onlinelibrary.wiley.com/doi/abs/10.1002/elps.201900200
    DOI

vir: Electrophoresis. - ISSN 0173-0835 (no. 516, Vol. 41, Apr. 2020, str. 386-393)
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