Screening and evaluation of endogenous reference genes for miRNA expression analysis in forensic body fluid samples

MicroRNA (miRNA)-based methods for body fluid identification are promising tools in the practice of forensic science. The selection of appropriate endogenous reference genes as normalizers for the relative quantification of miRNA expression levels using quantitative reverse transcription-polymerase chain reaction (RTqPCR) is essential to avoid errors and improve the comparability of miRNA expression level data among different body fluids. In this study, small RNAs were isolated from individual donations of five forensically relevant body fluids (peripheral blood, menstrual blood, saliva, semen and vaginal secretions). Thirty-seven samples were subjected to high-throughput miRNA sequencing. By combining our results with those obtained through a literature investigation, 28 candidate RNAs were identified. Following RTqPCR validation, the candidate RNAs were preliminarily evaluated in 15 samples to exclude miRNAs with low expression and high variation. Then, the expression levels of 10 relatively stable candidate reference RNAs in 100 samples were determined and further analysed using four commonly employed programs (geNorm, NormFinder, BestKeeper and ΔCq). According to the comprehensive stability rankings of the four algorithms, miR-320a-3p was validated as the most stable endogenous reference gene among the five forensically relevant body fluids, followed by miR-484, SNORD43, miR-320c and RNU6b. Moreover, the combined application of miR-320a-3p with RNU6b could increase the normalization effect. In addition, a total of 56 mock samples placed outdoors and indoors for different times were prepared to further evaluate the stability of candidate reference RNAs, and miR-320a-3p remained the preferred reference gene. Furthermore, the relative expression levels of publicly accepted body fluid-specific miRNAs were determined in 30 samples to verify the practicality and effectiveness of the reference genes. Our results revealed a set of alternative reference genes and could promote the development and application of miRNA-based body fluid identification by determining optional reference genes for strict normalization.