mRNA and microRNA stability validation of blood samples under different environmental conditions

RNA molecules, including mRNAs and microRNAs (miRNAs), have been used for forensic body fluid identification. Specific body fluids present unique mRNA expression patterns, while miRNAs identifying body fluids are mainly differentially expressed. miRNAs are thought to be more stable than mRNAs, although this lacks adequate supporting data. In this study, we addressed perceived concerns regarding the stability of miRNAs and mRNAs in blood samples. The samples used in this study involved three groups. First, environmentally-degraded blood stain samples were exposed to a range of environmental conditions over 1-360 days to degrade naturally. Second, simulated-degraded samples were prepared using RNase A or high temperature (80 °C). Furthermore, two authentic casework samples that were proven to be degraded from short tandem repeat (STR) profiles were analyzed. mRNAs and miRNAs present in the same blood samples were simultaneously detected through reverse transcriptase qPCR (RT-qPCR). Furthermore, mRNAs expression was determined by an mRNA multiplex PCR system. Our results showed that both mRNAs and miRNAs were stable in dry environments. The stability of miRNAs was relatively higher than that of mRNAs in humid environments or at high temperature. RNase A had the most serious impact on RNA stability, both mRNA profiles and miRNAs expression patterns were altered. The results of this study provide data and support to demonstrate that miRNAs represent more stable RNA molecules in body fluid identification compared to mRNAs.