Evaluating an mRNA based body fluid identification test using SYBR green fluorescent dye and real-time PCR

Abstract

The requirement to have more definitive and wider ranging body fluid identification (BFID) tests has resulted in a range of mRNA based real-time PCR BFI assays utilising Taqman fluorescent dye. An attempt to make a reliable and cost effective BFI test utilising the alternative SYBR Green fluorescent dye was carried out. RNA was extracted from blood and saliva stains from both male and female donors, this was then reverse transcribed using M-MLV and random hexamers. Using real-time PCR, relative quantitation of blood and saliva specific markers was carried out on the cDNA from the blood and saliva samples using the SYBR® Green fluorescent dye. Melting curve analysis was also performed immediately following PCR amplification. The relative quantitation values were calculated using the formula 2-ΔΔCT and all samples were normalised to reference gene 18s rRNA. The results revealed good specificity for a number of markers using this chemistry, however some markers were undetected. Blood markers NCF2, SPTB, PBDG and saliva specific markers HTN3, SPRR1A, KRT4 and KRT13 were investigated. In the SYBR green studies, the most specific markers were NCF2, KRT4, KRT13 and SPRR1A, showing reproducible results in a number of studies. Blood marker SPTB also appeared to be specific to blood however the melt curve data for this marker in each study was questionable given the low melting temperature for the amplified products. Blood specific marker PBGD, and saliva specific marker HTN3 were not detected using SYBR Green and saliva marker STATH was detected however in each case appeared to be non-specific in nature when them melt curves were analysed. Analysis of the 18s rRNA Ct values showed a higher expression in saliva than in blood in almost all instances, this may be due to collection of a higher number of cells when using a buccal swab, coupled with the inability to accurately quantify the RNA extracts before reverse transcription. Taqman assays were run on all markers as an additional test, to compare with the SYBR green data. All markers except SPTB showed very good specificity for their respective body fluids. SPTB, like in the SYBR green studies was detected in blood more than saliva, however detection was never consistent in each sample. It can therefore be said that real-time PCR using SYBR Green dye was capable of identifying specific mRNA markers blood and saliva however, the lack of specificity for this type of assay makes its use as a routine identification of body fluids in forensic casework not suitable. The main aim of this study was to develop a more cost effective BFID and as such involved the use of SYBR Green as a cheaper alternative to TaqMan. However, throughout these studies, it appeared to be quite costly in terms of validating a SYBR Green experiment, as more reagents were required in the long run due to vast amount of no template controls required per experiment. It therefore would appear that while SYBR Green is cheaper to buy, the cost to validate these type of experiments can be quite high, due to the non-specific nature of the dye itself. The SYBR Green studies were also much more time consuming with regards to data interpretation as post analysis of the amplification plot and melt curves is a necessity with this detection chemistry to ensure successful interpretation of the data