title: Automation of DNA and miRNA co-extraction for miRNA-based identification of human body fluids and tissues.
abstract: In the last years, microRNA (miRNA) analysis came into focus in the field of forensic genetics. Yet, no standardized and recommendable protocols for co-isolation of miRNA and DNA from forensic relevant samples have been developed so far. Hence, this study evaluated the performance of an automated Maxwell® 16 System-based strategy (Promega) for co-extraction of DNA and miRNA from forensically relevant (blood and saliva) samples compared to (semi-)manual extraction methods. Three procedures were compared on the basis of recovered quantity of DNA and miRNA (as determined by real-time PCR and Bioanalyzer), miRNA profiling (shown by Cq values and extraction efficiency), STR profiles, duration, contamination risk and handling. All in all, the results highlight that the automated co-extraction procedure yielded the highest miRNA and DNA amounts from saliva and blood samples compared to both (semi-)manual protocols. Also, for aged and genuine samples of forensically relevant traces the miRNA and DNA yields were sufficient for subsequent downstream analysis. Furthermore, the strategy allows miRNA extraction only in cases where it is relevant to obtain additional information about the sample type. Besides, this system enables flexible sample throughput and labor-saving sample processing with reduced risk of cross-contamination.
authors.affiliation: Institute of Legal Medicine, Ulm University, Ulm, Germany. galina.kulstein@uniklinik-ulm.de.
authors.forename: Galina
authors.initials: G
authors.lastname: Kulstein
authors.affiliation: Institute of Pathology, Ulm University, Ulm, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Marienfeld
authors.affiliation: Institute of Legal Medicine, Ulm University, Ulm, Germany.
authors.forename: Erich
authors.initials: E
authors.lastname: Miltner
authors.affiliation: Institute of Legal Medicine, Ulm University, Ulm, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Wiegand
mesh_terms: D057205:Automation, Laboratory; D001769:Blood; D004247:DNA / Q000302:isolation & purification*; D053803:Forensic Genetics; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000302:isolation & purification*; D009928:Organ Specificity; D060888:Real-Time Polymerase Chain Reaction; D012463:Saliva / Q000737:chemistry*
chemical_list: D035683:MicroRNAs; D004247:DNA
keywords: Body fluid; DNA/RNA co-extraction; Forensic RNA analysis; MicroRNA; Tissue identification