Evidence based strategy for normalization of quantitative PCR data, in forensic miRNA-analysis

Abstract

Micro-RNA (miRNA) based analysis of body fluids and composition of complex crime stains has recently been introduced as a potential and powerful tool to forensic genetics. Analysis of miRNA analysis has several advantages over mRNA but reliable miRNA detection and quantification using quantitative PCR requires a solid and forensically relevant normalization strategy. In our study we evaluated a panel of 12 carefully selected reference genes for their suitability as endogenous controls in miRNA qPCR normalization in forensically relevant settings. We analyzed assay performances and variances in venous blood, semen, menstrual blood, saliva and vaginal secretion and mixtures thereof integrating highly standardized protocols with contemporary methodologies and included several well established computational algorithms.Based on these empirical results, we recommend normalization to the group of RNU24, RNU43, and RNU66, as this signature exhibits the most stable expression levels and the least expected variation among the evaluated candidate reference genes in forensically relevant body fluids. To account for the lack of consensus on how best to perform and interpret quantitative PCR experiments, our study's documentation is according to MIQE guidelines, defining the “minimum information for publication of quantitative real-time PCR experiments”.