A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces
Introduction
Next to DNA-typing results, knowledge about the cellular origin of crime-related biological stains can be of significant importance for the reconstruction of the events at a crime scene. Conventional methods of body fluid identification, like the tetramethylbenzidine test, hexagon-obti and RSID-blood for blood stains [1], [2], the PSA and semenogelin test for semen [3] and the amylase tests (Phadebas or RSID-saliva) for saliva [4] are protein or enzyme-based, presumptive in nature and not always human-specific. Most of these methods rely on a colour-forming reaction which can be difficult to interpret, especially when dealing with coloured extracts or samples containing very little amounts of target material.
Within forensic genetics, messenger RNAs (mRNAs) have increasingly gained popularity regarding their potential to distinguish between human body fluids and other forensically relevant tissues [5], [6], [7], [8], [9], [10]. Alternative methods for cell-typing include tissue-specific miRNAs, DNA methylation [11], [12] and microbial markers [13]. miRNAs are small (20–24 nucleotides) regulatory RNAs which are strongly associated with members of a class of proteins called Argonautes [14], which makes them very stable and advantageous when dealing with degraded forensic samples [15], [16]. Also epigenetic DNA methylation markers have been described that can differentiate between some tissue types [11], [12]. Both miRNA and DNA methylation markers seem promising, but still in its infancy as for instance more markers are needed to discriminate the forensic range of body fluids. The use of microbial markers has been suggested for the identification of especially vaginal mucosa [13], [17], [18], [19]. However, it is not yet established whether the same microbes also occur on skin surfaces that are in close proximity of, or in contact with the vaginal microbial flora, such as skin surfaces of the hands, groin or penis. For these reasons, we regard tissue-specific mRNA analysis as the most versatile cell-typing approach.
mRNA-profiling has evolved from a singleplex PCR technique to a multiplex RT-PCR platform, providing expression of data on multiple genes simultaneously. mRNA-profiling is readily combined with DNA-genotyping since RNA and DNA can be obtained from the exact same sample [6], [9], [20], [21]. The different multiplexes that have been developed include markers for venous blood, saliva, semen, vaginal epithelia and menstrual secretion [9], [10], [13], and their selection was mainly based on the function described in literature [9], [10] or the tissue-specific expression as reported in expression databases [22]. Dedicated whole-genome expression array analysis in samples from forensically relevant body fluids that were stored for various time intervals has previously shown to deliver stable mRNA markers useful for forensic tissue identification [23], [24]. Skin is an additional forensically important cell type. Recently three mRNA transcripts (LOR, CDSN and KRT9) were reported to show high expression in skin samples relative to other forensically relevant cell types [25]. The addition of skin markers to an RNA-based cell-typing multiplex would increase the practical forensic value of the assay for two reasons: (1) a more complete view on all cell types present in an evidentiary trace is established which is important because skin cells are expected to occur in many crime scene samples and (2) an indication for the presence of contact DNA can be obtained. The most often used method to show the presence of contact traces is through dactyloscopic fingerprint analysis but also other microscopical and immunocytological techniques [26] have been described which can identify skin cells. Fingerprint visualisation methods do not apply to all types of substrates, and some can have negative effects on the nucleic acids in the skin cells [27] while others can introduce contamination [28]. To efficiently and objectively assess the biological origin of a forensic evidentiary trace, a single profiling assay was developed that assays both the forensically relevant body fluids and skin cells.
Section snippets
Sample collection
Body fluids and tissues from eight individuals were collected with their informed consent. Ten, 5, 1 and 0.5 μL blood, semen and saliva were collected on cotton swabs (Deltalab, Barcelona, Spain). The 1/10, 1/20, 1/100 and 1/1000 body fluid dilutions were prepared in phosphate buffered saline. Blood was collected through a finger prick (Accu-chek, Softclix Pro, Roche Diagnostics GmbH, Germany). Vaginal mucosa and menstrual secretions (day two of the menstrual cycle) were collected on cotton
Performance of the DNA/RNA co-isolation protocol
To assess whether the co-isolation procedure performs as equal as the in-house standard DNA isolation protocol, eight volumes of blood, saliva and semen ranging from 10 to 0.001 μL were applied to a cotton swab, processed using the DNA/RNA co-isolation protocol and the in-house DNA isolation protocol. The recovered gDNA was quantified and subjected to standard NGM DNA-profiling. The minimum volumes at which full DNA profiles were obtained, with both protocols, were 0.1 μL for blood and semen and
Concluding remarks
In this study, we describe a procedure to assess an evidentiary trace for two aspects: (1) who is the donor and (2) what cell type is present. From one sample, DNA and RNA are separately extracted using an extraction method that isolates both high and low molecular weight RNA molecules. While DNA is used for conventional STR-profiling, RNA is used for determining the cellular origin. For cell-typing, we designed a 19-plex RNA assay and showed that it profiles forensically relevant body fluids
Acknowledgements
The authors are grateful to all volunteers who donated samples for this work and thank Ate Kloosterman for providing stored samples. We thank Rolla Voorhamme for critically reading the manuscript. Role of funding: This study was supported by a grant from the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO) within the framework of the Forensic Genomics Consortium Netherlands (FGCN).
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