RNA/DNA co-analysis from human skin and contact traces--results of a sixth collaborative EDNAP exercise.

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.

A collaborative European exercise on mRNA-based body fluid/skin typing and interpretation of DNA and RNA results.

The European Forensic Genetics Network of Excellence (EUROFORGEN-NoE) undertook a collaborative project on mRNA-based body fluid/skin typing and the interpretation of the resulting RNA and DNA data. Although both body fluids and skin are composed of a variety of cell types with different functions and gene expression profiles, we refer to the procedure as 'cell type inference'. Nine laboratories participated in the project and used a 20-marker multiplex to analyse samples that were centrally prepared and thoroughly tested prior to shipment. Specimens of increasing complexity were assessed that ranged from reference PCR products, cDNAs of indicated or unnamed cell type source(s), to challenging mock casework stains. From this specimen set, information on the overall sensitivity and specificity of the various markers was obtained. In addition, the reliability of a scoring system for inference of cell types was assessed. This scoring system builds on replicate RNA analyses and the ratio observed/possible peaks for each cell type [1]. The results of the exercise support the usefulness of this scoring system. When interpreting the data obtained from the analysis of the mock casework stains, the participating laboratories were asked to integrate the DNA and RNA results and associate donor and cell type where possible. A large variation for the integrated interpretations of the DNA and RNA data was obtained including correct interpretations. We infer that with expertise in analysing RNA profiles, clear guidelines for data interpretation and awareness regarding potential pitfalls in associating donors and cell types, mRNA-based cell type inference can be implemented for forensic casework.

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative EDNAP exercise.

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.

RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise.

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 µl saliva, 5-0.01 µl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 µl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.