Optimization of the use of PrepFiler Express™ Forensic DNA extraction kit lysis buffer discard for mRNA profiling
2021
Hamnevoll, Kari
mRNA profiling for body fluid or tissue type identification is able to provide circumstantial information alongside DNA profiles from biological material found at a crime scene. To solve the challenge of material limitation in body fluid identification, studies have successfully developed mRNA profiles of RNA extracted from lysis buffer discard of DNA-extraction. It would be interesting to examine whether this method is possible for other combinations of DNA- and RNA-isolation kits. Promising results were found for RNA quantity and quality measured in the DNA-extraction lysis buffer discard and DNA-eluate from body fluids extracted with the PrepFiler Express™ DNA Extraction system. The protocols of three RNA-isolation kits were optimized for extraction from Prepfiler™ lysis buffer discard, and the kits were compared in terms of RNA quantity and quality. The mirVana™ kit was found to be most suited for this purpose. Although the ReliaPrep™ RNA Miniprep kit had similar results for quantity and quality, a continuous problem was contamination of genomic DNA. The Direct-zol™ RNA Miniprep kit gave poor results and was not further examined. A preliminary singleplex of body fluid specific mRNA markers and housekeeping genes, using different PCR programs was performed on RNA extracted from pure body fluids, and products were separated by capillary electrophoresis. An overload of the system was observed for most of the body fluid markers. Primer tests should be rerun with less RNA, e.g. RNA extracted from lysis buffer discard instead of pure body fluids, before further optimization.
显示更多 [+] 显示较少 [-]