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

Abstract

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.

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

“…The sensitivity of housekeeping gene detection was in the same range as the body-fluid-specific markers [26,39,40] and STR analysis (Table S7), though slightly reduced in saliva and semen compared to the saliva and semen specific markers [40]. Two of the housekeeping genes tested in this study, B2M and UBC, proved to be quite sensitive and were detected in most body fluid stains (Table S8).…”

“…Co-extracted DNA from the same stain provided good-quality STR profiles. Together with the precedent RNA exercises [38][39][40], a set of reliable RNA markers is now available for the identification of the most common forensic body fluids, namely blood, saliva, semen, menstrual blood and vaginal secretion. A subsequent EDNAP mRNA exercise will include an evaluation of mRNA markers for the identification of skin.…”

“…The suitability of mRNA profiling assays has also been demonstrated for old and environmentally compromised samples [17,18,35]. Recently, three collaborative exercises were performed by the European DNA Profiling Group (EDNAP -http://www.isfg.org/EDNAP) in order to evaluate the robustness and reproducibility of mRNA profiling for blood, saliva and semen identification: (1) evaluation of three blood-specific markers (HBB, SPTB and PBGD) using singleplex reactions [38]; (2) evaluation of seven blood-specific markers using two multiplex systems, a 'high sensitivity' duplex (HBB, HBA) and a 'moderate sensitivity' pentaplex (ALAS2, CD3G, ANK1, PBGD and SPTB) [39] and (3) a saliva triplex including the markers HTN3, STATH and MUC7 and a semen pentaplex allowing the detection and differentiation of sperm (PRM1, PRM2) and seminal plasma (PSA, SEMG1 and TGM4), the latter of which is necessary for the identification of semen from azoospermic men [40]. Most laboratories, some of which had no prior experience with RNA, were able to successfully isolate and analyze RNA from the provided samples.…”

Neurofibromatosis Type 1 Gene Mutation Analysis Using Sequence Capture and High-throughput Sequencing

“…The quality of DNA was evaluated by running the samples on agarose gels, which showed single bands larger than 10 kb. Saliva samples have more commonly been used in forensic medicine as a source for DNA (31), and the use of saliva in high throughput sequencing has been elucidated in a recent publication (32). Although the NF1 mutation analysis method described here is not yet validated for clinical application, it paves the way for new approaches in NF1 mutation analysis.…”

An Investigation of Various Tissue Specific mRNA Markers in Body Fluid Identification

“…42 Semen lekelerinin zamana bağlı stabilitesinin araştırıldığı bir başka çalışmada ise 20 yıldan fazla süre derin dondurucuda bekletilmiş örneklerde sonuç alınabildiği belirtilmektedir. 43 Bu çalışmada farklı ortam koşullarında [6 ay, üç ayrı sıcaklıkta-oda sıcaklığı (22-25°C), etüv (37°C) ve buzdolabı (4°C)] bekletilmiş 5'er adet kan, menstrüel kan, semen, tükürük ve vajinal sekresyon lekeleri ile yapılan çalışmalarda tüm mRNA belirteçlerinin belirlenen sürelerde ve sıcaklıklarda tanımlanabildiği gözlenmiştir.…”