A collaborative European exercise on mRNA-based body fluid/skin typing and interpretation of DNA and RNA results

Abstract

The European Forensic Genetics Network of Excellence (EUROFORGEN-NoE) undertook a collaborative project on mRNA-based body fluid/skin typing and the interpretation of the resulting RNA and DNA data. Although both body fluids and skin are composed of a variety of cell types with different functions and gene expression profiles, we refer to the procedure as 'cell type inference'. Nine laboratories participated in the project and used a 20-marker multiplex to analyse samples that were centrally prepared and thoroughly tested prior to shipment. Specimens of increasing complexity were assessed that ranged from reference PCR products, cDNAs of indicated or unnamed cell type source(s), to challenging mock casework stains. From this specimen set, information on the overall sensitivity and specificity of the various markers was obtained. In addition, the reliability of a scoring system for inference of cell types was assessed. This scoring system builds on replicate RNA analyses and the ratio observed/possible peaks for each cell type [1]. The results of the exercise support the usefulness of this scoring system. When interpreting the data obtained from the analysis of the mock casework stains, the participating laboratories were asked to integrate the DNA and RNA results and associate donor and cell type where possible. A large variation for the integrated interpretations of the DNA and RNA data was obtained including correct interpretations. We infer that with expertise in analysing RNA profiles, clear guidelines for data interpretation and awareness regarding potential pitfalls in associating donors and cell types, mRNA-based cell type inference can be implemented for forensic casework.

Degraded RNA transcript stable regions (StaRs) as targets for enhanced forensic RNA body fluid identification

“…The use of RT-PCR to detect mRNA transcripts has shown that inconsistent results can be observed and explained as 'sporadic' in some studies [38]. To help explain these findings, we used MPS data from fresh and degraded body fluids mapped against the human reference genome (hg19) to deliberately design primers for mRNA regions in which higher read counts were observed.…”

“…Further testing was undertaken to assess the reproducibility of target detection using StaR primers, as the inconsistency of RNA marker detection could be problematic in forensic body fluid identification [38]. Replicate amplifications using conventional HTN3, UBE2D2, and SLC4A1 primers resulted in irregular (or inconsistent) target amplification and some replicates failed to generate amplicons (Figs.…”

“…Our new StaR primer design concept to target the stable regions of RNA transcripts demonstrates the possibility for consistent RNA marker and body fluid identification when conventional primers would have failed. The inconsistency of RNA marker analysis in degraded forensic body fluids is an on-going problem [38] but we believe that our StaR primer design approach is a significant breakthrough to address this problem. The StaR concept is a fundamental new development in RNA analysis technology that we believe is applicable to fields beyond forensic science.…”

Highly degraded RNA can still provide molecular information: An in vitro approach

“…In Forensics, there has been an increase in interest for RNA studies. Out of the several fields of application , the tissue‐specific gene expression of RNA makes this molecule the ideal tool for determining the cellular origin of unknown samples . In addition, most recently, tissue‐specific transcriptional changes were described as a response to the death of the human organism, thus opening the frontiers to a molecular evaluation of the postmortem interval .…”

Differentially methylated embryonal Fyn-associated substrate (EFS) gene as a blood-specific epigenetic marker and its potential application in forensic casework

“…For example, current techniques would not normally distinguish venous from menstrual blood. While both would give positive reactions using presumptive chemical tests, even tissue-specific mRNA typing could still raise doubts about the origin, mainly due to the co-existence of blood peaks when menstrual blood is present [23,24]. In DNA methylation-based studies in forensic identification for blood, potential markers such as cg06379435 [25,26], cg08792630 [26], cg26285698 [12], cg04011671 and cg18454288 [14] have been identified through genome-wide methylation arrays, such as the Illumina Human Methylation 450K bead array.…”