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In recent years, forensic mRNA profiling has increasingly been used to identify the origin of human body fluids. By now, several laboratories have implemented mRNA profiling and also use it in criminal casework. In 2018 the FoRNAP (Forensic RNA Profiling) group was established among a number of these laboratories with the aim of sharing experiences, discussing optimization potential, identifying challenges and suggesting solutions with regards to mRNA profiling and casework. To compare mRNA profiling methods and results a collaborative exercise was organized within the FoRNAP group. Seven laboratories from four countries received 16 stains, comprising six pure body fluid / tissue stains and ten mock casework samples. The laboratories were asked to analyze the provided stains with their in-house method (PCR/CE or MPS) and markers of choice. Five laboratories used a DNA/RNA co-extraction strategy. Overall, up to 11 mRNA markers per body fluid were analyzed. We found that mRNA profiling using different extraction and analysis methods as well as different multiplexes can be applied to casework-like samples. In general, high input samples were typed with high accuracy by all laboratories, regardless of the method used. Irrespective of the analysis strategy, samples of low input or mixed stains were more challenging to analyze and interpret since, alike to DNA profiling, a higher number of markers dropped out and/or additional unexpected markers not consistent with the cell type in question were detected. It could be shown that a plethora of different but valid analysis and interpretation strategies exist and are successfully applied in the Forensic Genetics community. Nevertheless, efforts aiming at optimizing and harmonizing interpretation approaches in order to achieve a higher consistency between laboratories might be desirable in the future. The simultaneous extraction of DNA alongside RNA showed to be an effective approach to identify not only the body fluid present but also to identify the donor(s) of the stain. This allows investigators to gain valuable information about the origin of crime scene samples and the course of events in a crime case.

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.

The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent. Fil: Corach, Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Caputo, Mariela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marino, Miguel Eduardo. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Analisis de ADN; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Purps, Josephine. Charité-Universitätsmedizin; Alemania Fil: Siegert, Sabine. University of Cologne; Alemania Fil: Willuweit, Sascha. Charité-Universitätsmedizin; Alemania Fil: Nagy, Marion. Charité-Universitätsmedizin; Alemania Fil: Alves, Cíntia. Universidad de Porto; Portugal Fil: Salazar, Renato. Universidad de Porto; Portugal Fil: Angustia, Sheila M. T.. Philippine National Police Crime Laboratory; Filipinas Fil: Santos, Lorna H.. Philippine National Police Crime Laboratory; Filipinas Fil: Anslinger, Katja. Universitat Genzentrum Der Ludwing-maximilians; Alemania Fil: Bayer, Birgit. Universitat Genzentrum Der Ludwing-maximilians; Alemania Fil: Ayub, Qasim. The Wellcome Trust Sanger Institute; Reino Unido Fil: Wei, Wei. The Wellcome Trust Sanger Institute; Reino Unido Fil: Xue, Yali. The Wellcome Trust Sanger Institute; Reino Unido Fil: Tyler Smith, Chris. The Wellcome Trust Sanger Institute; Reino Unido Fil: Baeta Bafalluy, Miriam. Universidad de Zaragoza; España Fil: Martínez Jarreta, Begoña. Universidad de Zaragoza; España Fil: Egyed, Balazs. Eotvos University, Budapest; Argentina Fil: Balitzki, Beate. Universidad de Basilea; Suiza Fil: Tschumi, Sibylle. Universidad de Basilea; Suiza Fil: Ballard, David. King; Reino Unido Fil: Syndercombe Court, Denise. King; Reino Unido Fil: Barrantes, Xinia. Poder Judicial, Forensic Sciences Department; Costa Rica Fil: Bäßler, Gerhard. Landeskriminalamt Baden-Württemberg; Alemania Fil: Berger, Burkhard. Universidad de Innsbruck; Austria Fil: Niederstätter, Haral. Universidad de Innsbruck; Austria Fil: Parson, Walther. Universidad de Innsbruck; Austria. University Park; Estados Unidos Fil: Davis, Carey. Department of Molecular and Medical Genetics; Estados Unidos. Institute of Applied Genetics; Estados Unidos Fil: Furfuro, Sandra. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; Argentina Fil: Locarno, Laura. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; Argentina