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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.

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology. © 2011 Elsevier Ireland Ltd. All rights reserved.

17 Y-chromosome STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS456, DYS391, DYS392, DYS393, DYS385 a/b, DYS458, DYS439, DYS635, GATA H4.1, DYS437, DYS438 and DYS448) were determined for 303 unrelated males, living in Central Portugal, using the AmpFlSTR ® YFiler™ PCR Amplification kit (Applied Biosystems). A total of 287 different haplotypes were found, 272 being unique. The overall haplotype diversity (HD) was determined as 0.9996, a value similar to other YFiler data sets. Y-STR polymorphisms in Central Portugal population, using YFiler, provide a powerful discrimination tool for routine forensic applications.

The European DNA Profiling (EDNAP) Group performed a collaborative exercise on a mitochondrial (mt) DNA screening assay that targeted 16 nucleotide positions in the coding region and allowed for the discrimination of major west Eurasian mtDNA haplogroups. The purpose of the exercise was to evaluate the stability and reproducibility of the self-developed multiplex-PCR and multiplex-single base extension kit by blind-testing saliva and hair shaft samples provided by the organizing laboratory. The overall success rate in obtaining useful results was high given that some of the participating laboratories had no previous experience with the technology and/or mtDNA analysis. The results of this collaborative exercise stimulate the expansion of screening methods in forensic laboratories to increase efficiency and performance of mtDNA typing, and thus demonstrates that mtDNA SNP typing is a powerful tool for forensic casework analysis.