title: An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensically relevant body fluids.
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 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 13 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, saliva, semen, menstrual blood, 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 SNORD24, SNORD38B, and SNORD43 as this signature exhibits the most stable expression levels and the least expected variation among the evaluated candidate reference genes in the given set of 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 compliant to MIQE guidelines, defining the "minimum information for publication of quantitative real-time PCR experiments".
authors.affiliation: Institute of Legal Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Sauer
authors.affiliation: Institute of Legal Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Madea
authors.affiliation: Institute of Legal Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany. Electronic address: cornelius.courts@uni-bonn.de.
authors.forename: Cornelius
authors.initials: C
authors.lastname: Courts
mesh_terms: D001826:Body Fluids / Q000378:metabolism*; D053803:Forensic Genetics; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D035683:MicroRNAs
keywords: Body fluid identification; Endogenous references; Micro-RNA; Normalization; RT-qPCR