Abstract：
RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 μg/μL - 0.24 ng/μL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 μL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments.
摘要：
RNA分析在法医学中显示出巨大的价值,如体液和组织鉴定，死后间隔估计，生物年龄预测,等。目前,大多数RNA后续实验涉及逆转录（RT）程序。已经表明，RT步骤是可变的，并且对后续的数据分析有较大的影响，尤其是法医痕迹样本.然而,不同RNA模板输入和互补DNA（cDNA）产量之间的变异模式尚不清楚。在这项研究中,一系列2倍梯度稀释的RNA标准品（1μg/μL-0.24ng/μL）和法医样本（包括血液样本，唾液样本,血迹,和唾液染色）使用EasyQuickRTMasterMix进行逆转录。通过液滴数字PCR(ddPCR)定量获得的cDNA以评估ACTB基因的RT产率。结果表明，在所选试剂盒的10μLRT反应系统中，125ngRNA模板的RT产率最高。对于所有染色样品，由于RNA数量低于125ng，因此RT产量随着RNA模板输入量的增加而提高。由于许多使用不同种类酶的商业化逆转录试剂盒可用于法医RNA研究，我们建议在使用任何试剂盒进行逆转录实验时,应提前进行系统实验,以确定最佳RT产量时的RNA输入量.
