Effetti dei metodi di prelievo e conservazione di tracce su indagini forensi di DNA e mRNA

The increasing sensitivity of multiplex PCR assays for the simultaneous amplification of short tandem repeat (STR) loci allows forensic investigators to recover DNA profiles from minute biological samples found at the crime scene (so called "trace DNA"). Determination of tissue origin of trace DNA is often crucial for the reconstruction of crime dynamics. Recently, several robust protocols for DNA/RNA co-extraction from stains have been described and validated for forensic purposes, thus enabling the single pipeline analysis of both STRs and mRNA profiles to identify body fluids. So far, limited research was dedicated to the identification of optimal methods for the retrieval and preservation of RNA from forensic stains. Storage conditions of trace collection swabs may also affect the quantity and quality of retrievable nucleic acids. The goal of this work was to evaluate the effects of different swabbing methods, and subsequent storage conditions, on DNA/mRNA profiling of trace evidence. Mock stains were created by applying to clean glass slides different body fluids/tissues of forensic interest obtained from a single donor (whole blood, 1:10 diluted blood treated with luminol, saliva, semen, skin deposited by rubbing of the thumb/index fingertip). 24 traces per tissues, for a total of 120 samples, were deposited. Traces were collected 3 days after deposition, using sterile cotton swabs moistened with: Rnase-free water; absolute ethanol; a commercial RNA stabilizing solution (RNAlater®). Swabs were either dried at room temperature or freezed for 1/7 days before extraction. After DNA/RNA co-isolation, total RNA was quantified and mRNA underwent reverse transcription followed by multiplex PCR profiling of a panel of 19 tissue-specific and housekeeping genes. Genomic DNA was also quantified and subjected to STR profiling. STR typing results were compared to those obtained from a reference buccal swab of the donor. Concentration of total RNA isolated from mock stains was significantly higher for swabs treated with RNAlater® compared with water (p<0.001) and ethanol (p<0.001), with consequent effects on mRNA profiling. In 95% of traces collected with RNAlater®, the expected combination of tissue-specific gene markers was observed, based on mRNA profiling results, compared to 85% for ethanol, and 67.5% for water. Interestingly, the impact of moistening media on DNA recovery rates varied among tissues. In skin samples, both ethanol (p<0.001) and RNA stabilizer (p<0.05) significantly outperformed water, also generating more complete STR profiles. On the contrary, DNA retrieval was significantly lower for ethanol and RNAlater® in whole blood (p<0.001), and ethanol (p<0.05) in semen. However, because of the higher DNA yields obtained from whole blood compared to skin, moistening agents had no effective impact on the quality of STR typing in this case. Finally, no significant differences in DNA/RNA recovery were seen depending on storage conditions and storage time of collection swabs. The obtained results demonstrated that alternative moistening media for collection swabs, like ethanol and a RNA stabilizer, significantly improve mRNA analysis in forensic stains, without compromising STR typing. Moreover, in a lipidic-rich tissue as skin, they enhance DNA recovery and profiling rates, compared to water.