Department of Medicine, Surgery and Health, University of Trieste, Trieste, Italy.
Department of Drug Sciences, University of Pavia, Pavia, Italy.
Electrophoresis. 2020 Mar;41(5-6):386-393. doi: 10.1002/elps.201900200. Epub 2020 Feb 3.
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 (r > 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.
RNA 在死后组织中的长期存活是一个棘手的问题。许多陈旧/法医标本实际上显示出基于 PCR 的高比例无效/不确定结果，而可靠的结果有时则来自考古样本。另一方面，有几项数据表明，RNA 是一种能够耐受多种物理化学压力的分子。在本研究中，一种已开发用于长时间水解 DNA 的简单方案被应用于从血液中提取的 RNA 样本。该方案基于在 70°C 下使用超纯水和二乙基焦碳酸酯水作为水解介质进行长达 36 小时的热介导（70°C）水解。即使微流控装置显示出渐进式降解模式，也未发现脱嘌呤水平。对两个（60bp 长）管家基因靶标（甘油醛-3-磷酸脱氢酶和卟胆原脱氨酶）的反转录/定量 PCR 分析表明，可扩增靶标（%AT）的百分比随损伤处理时间的延长而降低（r > 0.973）。与经历相同损伤处理的 DNA 样本相比，降解 RNA 中的%AT 表明，%AT 始终更高，最高可达三个数量级。最后，即使终点 PCR 分析血液特异性标志物也能给出可靠的结果，这与样本的体液来源一致。总之，所有基于 PCR 的结果表明，RNA 在 70°C 下在温和酸性缓冲液中孵育 36 小时后，仍然能够将逆转录为短 cDNA 片段。因此，RNA 样本的长期存活可能主要取决于对 RNA 酶攻击的保护，而不是对环境因素（如湿度和酸度）的稳定性，这些因素对 DNA 的稳定性非常重要。最后，我们的结果表明，即使 DNA 图谱分析失败，RNA 分析也可以成功进行。
