title: An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensic organ tissue identification.
abstract: Messenger-RNA (mRNA)-based analysis of organ tissues and their differentiation in complex crime stains has recently been introduced as a potential and powerful tool to forensic genetics. Given the notoriously low quality of many forensic samples it seems advisable, though, to substitute mRNA with micro-RNA (miRNA) which is much less susceptible to degradation. However, reliable miRNA detection and quantification using quantitative PCR requires a solid and forensically relevant normalization strategy. In our study we evaluated a panel of 15 carefully selected reference genes for their suitability as endogenous controls in miRNA qPCR normalization in forensically relevant settings. We analyzed assay performances and expression variances in 35 individual samples and mixtures thereof integrating highly standardized protocols with contemporary methodologies and included several well-established computational algorithms. Based on these empirical results, we recommend SNORD48, SNORD24, and RNU6-2 as endogenous references since these exhibit the most stable expression levels and the least expected variation among the evaluated candidate reference genes in the given set of forensically relevant organ tissues including skin. 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".
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: Iris
authors.initials: I
authors.lastname: Babion
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: D000293:Adolescent; D000328:Adult; D000368:Aged; D001921:Brain / Q000378:metabolism; D002648:Child; D005260:Female; D053803:Forensic Genetics; D019983:Guideline Adherence; D017408:Guidelines as Topic; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000378:metabolism; D055815:Young Adult
chemical_list: D035683:MicroRNAs
keywords: Endogenous references; Normalization; Organ tissue identification; RT-qPCR; micro-RNA