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Evaluation of the Effects of Different Sample Collection Strategies on DNA/RNA Co-Analysis of Forensic Stains.

Author(s): Lacerenza D, Caudullo G, Chierto E, Aneli S, Di Vella G, Barberis M, Voyron S, Berchialla P, Robino C

Publication: Genes (Basel), 2022, Vol. 13, Page

PubMed ID: 35741745 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the potential effects of the fluid type used to moisten swabs for collection of blood, luminol-treated diluted blood, semen, saliva and skin specimens from glass slides on the DNA and RNA yield, the ability to obtain a complete STR profile, and the proper classification of specimen type based on mRNA profile. The effects of swab storage temperature and duration were also investigated. Whole blood, semen, and saliva as well as blood that was diluted 1:10 with water were obtained from a single male donor (age 33); 10 µL aliquots were applied to replicate slides (24 for each fluid type). Skin specimens (24 total) were obtained from the same donor by rubbing the thumb and index finger over the glass slide for 10 sec, 1 h after hand washing. Slides were stored for 3 days at room temperature (15.5-24°C, <60% humidity). Slides containing diluted blood were treated with a luminol solution just before specimen collection. Specimens were collected from each slide using sterile swabs that had been moistened with 20 µL RNase-free water, absolute anhydrous ethanol, or RNAlater. Swabs were then stored at room temperature or frozen at -20°C for 1 or 7 days before DNA/RNA isolation. Swabs were incubated in RLT buffer with carrier RNA at 56°C for 3 h before DNA and RNA isolation using the AllPrep DNA/RNA Micro Kit. Genomic DNA was collected from buccal cells of the donor using the ChargeSwitch gDNA Normalized Buccal Cell Kit. DNA genotyping was performed using the AmpFlSTR® Identifiler® Plus Kit and detected by capillary electrophoresis using a 3500 Genetic Analyzer. RNA was reverse transcribed using RETROscript Kit and levels of three blood markers (ALAS2, CD93,  and HBB), two saliva markers (HTN3 and STATH), two nasal mucosal markers (STATH and BPIFA1), three semen markers (KLK3, SEMG1, and PRM1), three vaginal mucosa markers (CYP2B7P1, MUC4, and MYOZ1), three markers of menstrual secretions (MMP7, MMP10, MMP11), two skin markers (CDSN and LCE1C), and two housekeeping genes (ACTB and 18S-rRNA) were amplified by real-time PCR and detected by 3500 Genetic Analyzer.

   Summary of Findings:

   The optimal cDNA input (no saturated peaks) was experimentally determined to be 3.5 µL for all of the specimen types except semen, which had an optimal volume of 1 µL. DNA recovery from blood, semen and skin specimens was significantly affected by the fluid used to moisten the swabs. DNA yields from blood and semen were significantly higher when water was used rather than ethanol (P<0.001 and P=0.029), but DNA yield using water was only non-significantly higher than when RNAlater was used. DNA yields from skin were significantly higher when ethanol was used rather than water (P=0.014). While not significant, DNA yields from luminol-treated diluted blood and saliva were highest when RNAlater was used, followed by water.  Storing swabs at -20°C rather than room temperature resulted in higher yields of DNA from luminol-treated diluted blood (P=0.019) and saliva (P<0.001), but had no effect on DNA yields from blood, semen or skin. DNA yield was not significantly different in swabs stored for 1 day versus 7 days.

    

   RNA yields from all specimens, except for saliva, were significantly affected by the type of fluid used to moisten the swab and were highest when swabs were moistened with RNAlater. RNA yields from saliva, while not significantly affected by fluid type, were highest when swabs were moistened with water.  RNA yield was not significantly affected by storage of the swabs at -20°C rather than at room temperature but was higher when swabs from saliva were stored for 1 day than 7 days (P<0.001) and swabs from semen were stored for 7 days versus 1 day (P=0.045).

   All specimens yielded full STR profiles regardless of the type of fluid used to moisten the swab and swab storage, except for skin specimens. For skin specimens, the STR profile was more complete when compared to the buccal cell profile when ethanol (99.6%) or RNAlater (98.7%) were used to moisten the swab than when water was used (71%; P=0.006 versus ethanol and P=0.018 versus RNA later). The allelic imbalance when water was used to moisten the swab was particularly evident for amplicons >150 bp. The fluid used to moisten the swab significantly affected the percentage of blood specimens properly classified based on mRNA profile, with 100% correctly classified when ethanol or RNAlater was used and only 37.5% when water was used (P=0.04, both). While not significant, 100% proper classification based on mRNA profile was achieved using water for luminol-treated blood; using ethanol for blood, luminoltreated blood, and semen; and using RNAlater for blood, semen and skin specimens. Absolute (100%) proper classification was not achieved for saliva specimens, where 87.5% were properly classified when RNAlater was used to moisten swabs, 50% when ethanol was used, and 37.5% when water was used. In general, proper classification based on mRNA profile was lower when swabs were stored 7 days versus 1 day, but the difference was only significant for saliva (91.7% versus 25%, P=0.003). For skin specimens, a higher percentage of swabs were properly classified when they were stored for 7 days compared to those stored for 1 day (91.7% versus 75%, not significant). While storing swabs at -20C led to a higher percentage of properly classified specimens compared to when swabs were stored at room temperature for semen specimens, no significant differences of storage temperature were observed among the other specimen types evaluated. The authors report that, in general, swabs moistened with RNAlater and stored at -20°C had the highest PCR signal, but significance was dependent on specimen type.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - Skin
   • Bodily Fluid - Semen
   • Bodily Fluid - Saliva

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Blood Saliva Skin Semen Luminol treated blood
   Storage	Storage temperature	-20°C Room-temperature
   Storage	Storage duration	1 day 7 days
   Storage	Short-term storage solution	Water Ethanol RNAlater

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