A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces

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Abstract

In current forensic practice, information about the possible biological origin of forensic traces is mostly determined using protein-based presumptive testing. Recently, messenger RNA-profiling has emerged as an alternative strategy to examine the biological origin. Here we describe the development of a single multiplex mRNA-based system for the discrimination of the most common forensic body fluids as well as skin cells. A DNA/RNA co-isolation protocol was established that results in DNA yields equivalent to our standard in-house validated DNA extraction procedure which uses silica-based columns. An endpoint RT-PCR assay was developed that simultaneously amplifies 19 (m)RNA markers. This multiplex assay analyses three housekeeping, three blood, two saliva, two semen, two menstrual secretion, two vaginal mucosa, three general mucosa and two skin markers. The assay has good sensitivity as full RNA profiles for blood, semen and saliva were obtained when using ≥0.05 μL body fluid starting material whereas full DNA profiles were obtained with ≥0.1 μL. We investigated the specificity of the markers by analysing 15 different sets of each type of body fluid and skin with each set consisting of 8 individuals. Since skin markers have not been incorporated in multiplex endpoint PCR assays previously, we analysed these markers in more detail. Interestingly, both skin markers gave a positive result in samplings of the hands, feet, back and lips but negative in tongue samplings. Positive identification (regarding both DNA- and RNA-profiling) was obtained for specimens stored for many years, e.g. blood (28 years-old), semen (28 years-old), saliva (6 years-old), skin (10 years-old) and menstrual secretion (4 years-old).

The described approach of combined DNA- and RNA-profiling of body fluids and contact traces assists in the interpretation of forensic stains by providing information about not only the donor(s) that contributed to the stain but also by indicating which cell types are present.

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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