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 Table of Contents  
CASE REPORT
Year : 2022  |  Volume : 8  |  Issue : 3  |  Page : 118-122

Body fluid identification by messenger RNA profiling in sexual assault


Division of Forensic Genetics, Key Laboratory of Forensic Genetics, Institute of Forensic Science, Ministry of Public Security, Beijing, China

Date of Submission15-Aug-2021
Date of Decision24-Apr-2022
Date of Acceptance29-May-2022
Date of Web Publication02-Sep-2022

Correspondence Address:
Wanshui Li
Institute of Forensic Science, Ministry of Public Security 17# Muxidi Nanli, Xicheng District, Beijing 100038
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jfsm.jfsm_54_21

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  Abstract 


Body fluid identification through messenger RNA (mRNA) has been proposed as a useful supplement to presumptive and confirmatory tests by previous laboratory studies; however, its application in routine clinical forensic examination was rare. We report a case of sexual assault in which body fluid identification by mRNA profiling was used. Vaginal secretions mRNA markers (MUC4, HBD1, and CYP2B7P1) were used to test the sample, being obtained positive results. This case demonstrates that mRNA profiling of body fluids could be applied to routine case examinations as an aid, acting as a scientific collaborative evidence to strengthen the medicolegal opinion.

Keywords: Body fluid identification, messenger RNA profiling, sexual assault


How to cite this article:
Wang C, Zhao H, Meng Q, Sun H, Xu X, Li W. Body fluid identification by messenger RNA profiling in sexual assault. J Forensic Sci Med 2022;8:118-22

How to cite this URL:
Wang C, Zhao H, Meng Q, Sun H, Xu X, Li W. Body fluid identification by messenger RNA profiling in sexual assault. J Forensic Sci Med [serial online] 2022 [cited 2022 Sep 27];8:118-22. Available from: https://www.jfsmonline.com/text.asp?2022/8/3/118/355566




  Introduction Top


Test and identification of body fluid stains from crime scene constitutes an important part in forensic practice. Such tests aim not only to analyze DNA and identify its host but also to find out the source tissue of the fluid. However, in view of the fact that conventional body fluid identification methods may have a high false-positive rate,[1] may be destructive to the sample, and cannot be applied in some body fluids, researchers have tried many new approaches.[2] These methods provide new technical support for body fluid identification from different angles.

Among these new approaches, messenger RNA (mRNA) profiling is the most frequently used approach to determine the type of unknown body fluid stain or to discriminate between different body fluids.[3],[4],[5] Previous studies have demonstrated the existence of several tissue-specific mRNA markers and reported polymerase chain reaction (PCR) platforms to distinguish body fluids in reference samples or mock case samples, but few reported real case examinations. Here, we report a case of sexual assault where we successfully detected the specific mRNA markers of vaginal secretions from the evidence, which provided key evidence for the litigation of the case.


  Materials and Methods Top


Case information

A disputed sexual assault case was submitted to our laboratory to confirm a rape crime. A young woman claimed that she has been sexually assaulted, and the suspect used a condom. However, the suspect argued that the condom was just touched by the victim. Full short tandem repeat (STR) profiles were obtained both from the victim and the suspect. The local police department wanted to determine the fluid origin of the victim's STR profile, determining whether this condom was really inserted into the victim's vagina or if the victim's DNA was only transferred to the condom by hand contact. Therefore, either acquitting or convicting the suspect of a sexual assault claim. Our laboratory reported mRNA analysis to solve this problem.

Case sample

Four samples were collected by criminal technicians of local public security bureau in this case. Sample 1 was the victim's vaginal swab. Sample 2 was a condom provided by the victim to the possible suspect. Sample 3 was the victim's oral swab, and sample 4 was the victim's hand swab. Samples 1, 3, and 4 were taken by physical examination. All the samples were stored at −80°C before RNA and DNA extraction. The collection procedure was approved by the Human Ethics Committee of the Institute of Forensic Science, Ministry of Public Security (Approval No. 20200003, approved on March 15, 2020).

RNA and DNA extraction and quantification

Swab samples were cut into pieces before extraction. The condom sample was wiped by a cotton swab from the outside surface (dipped by Buffer RLT from kit) and then cut the swab into pieces for extraction. Total RNA was extracted by RNeasy Micro Kit (QIAGEN, Germany) according to its manufacturers' recommended procedures with the following adaptions: (1). All the samples were treated with β-mercaptoethanol (1%, added in 350 μl Buffer RLT) and then incubated at 4°C overnight. (2) In RNeasy Micro Kit, the follow-through liquid was collected to extract DNA using MagAttract M48 DNA Manual Kit (Qiagen). RNA and DNA were quantified by Qubit 3.0 Fluorometer according to the manufacturers' recommendations. To get as much potential body fluids as possible and due to the elution volume limitation of the RNeasy kit, when extracting samples, we used multiple cotton swabs to wipe and performed RNA extraction and reverse transcription (RT), respectively.

Reverse transcription

RT was carried out by SuperScript III First-Strand Synthesis System (Life Technologies, USA) according to the manufacturers' recommendations. Due to the low RNA content of the sample samples found in daily experiments, the maximum RNA input was used to ensure successful RT. The reaction volume was 21 μl, contains 8 μl RNA template, 2 μl 10 × RT-Buffer, 1 μl random hexamers (2.5 mM), 1 μl dNTP (2.5 mM), 4 μl Mg2+ (25 mM), 2 μl Dithiothreitol (DTT) (0.1 M), 1 μl SSIII reverse transcriptase (200 U/μl), 1 μl RNaseOUT (40 U/μl), and 1 μl RNase H (2 U/μl). RT-control was added. The cDNA was stored at − 80°C before PCR.

Endpoint polymerase chain reaction

Primers for MUC4, CYP2B7P1, HBD1, STATH, PRM2, TGM4, GlycoA, SPTB, MMP7, and B2M were used [Invitrogen, [Table 1]]. Multiple cDNA templates of the same sample were combined during PCR amplification. Each singleplex PCR reaction contained 5 μl cDNA template, 14 μl water (diethyl pyrocarbonate [DEPC] treated), 2 μl primer (10 mM), 2 μl 10 × PCR Buffer (Applied Biosystems, USA), 1.25 μl dNTP Mix (Takara, China), 2.5 μl Mg2+ (Applied Biosystems), and 0.25 μl Gold Taq Polymerase (Applied Biosystems). Negative control was used (DEPC-treated water), and positive control was used (the victim's vaginal swab).
Table 1: Primer sequences in RNA amplification

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All the samples were amplificated twice, and the “x ≥ n/2” principle[12] was followed to explain the result.

PCR reaction was performed on a GeneAmp 9700 PCR System (Applied Biosystems) with the following conditions: initial denaturation at 95°C for 15 min, 40 cycles of 94°C for 20 s, 60°C for 30 s, 72°C for 40 s, and a final extension at 72°C for 10 min.

Short tandem repeat profiling

DNA samples were amplified using AmpFlSTR GlobalFiler PCR Amplification Kit (Applied Biosystems) with the following conditions: initial denaturation at 95°C for 1 min, 29 cycles of 94°C for 10 s, 59°C for 90 s, and a final extension at 60°C for 10 min.

To confirm the suspect, extracted DNA of the sample was amplified by AmpFlSTR Y-Plus Amplification Kit (Applied Biosystems) with the following conditions: 95°C for 1 min, 29 cycles of 94°C for 4s, 61.5°C for 60 s, and a final extension at 60°C for 22 min.

Capillary electrophoresis

Capillary electrophoresis was performed on a 3500XL Genetic Analyzer (Applied Biosystems). Profiles were analyzed by GeneMapper ID-X v1.4 (Applied Biosystems). The threshold was set to 150 RFU for both DNA and RNA results.


  Results Top


Capillary electrophoresis

In this case sample, ten mRNA markers (MUC4, CYP2B7P1, HBD1, STATH, PRM2, TGM4, GlycoA, SPTB, MMP7, and B2M) were analyzed [Table 2]. The mRNA markers of MUC4, CYP2B7P1, HBD1, B2M, PRM2, and TGM4 presented positive results, suggesting that the surface of the condom contained both vaginal secretions and semen [Figure 1] and [Figure 2]. This result pointed out to confirm that the analyzed condom was inserted into the victim's vagina.
Table 2: CE results for samples 1-4

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Figure 1: Electropherograms for sample 2

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Figure 2: Electropherogram of CYP2B7P1 from sample 1

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Short tandem repeat profiling

STR profiling got from samples 1, 3, and 4 were all the victim's single DNA profile, STR profiling got from sample 2 was a mixed DNA profile combined by the victim and the suspect [Figure 3]. Y-STR showed that sample 2 got a full Y-genotype, which confirmed the suspect's genotype [Figure 4]. There is no significant difference between the co-extracted test results and conventionally extracted DNA.
Figure 3: The genotype of sample 2: A mix of the victim and the suspect

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Figure 4: Y-STR of sample 2: A full genotype of the suspect. Y-STR: Y-short tandem repeat

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  Discussion Top


Messenger RNA profiling in real case investigation

Body fluid stain identification has played an important role in criminal case investigation and court judgment in recent years. The prosecute and defense sides in court both start to focus on the source of DNA rather than whether the DNA comes from the suspect, which means how the DNA is there. Even if in the cases that DNA gets matched, the police tend to exert more effort in body fluid stain identification. According to the case assessment and interpretation model, only subsource-level DNA evidence is not enough, and the source-level evidence can give more information and is more powerful to prove the suspect's activity.[13] The effectiveness of conventional methods in this field is often compromised by their high rate of false results, possible damage on the test materials, and inability of testing body fluid of certain kinds. To tackle these problems, attempts were made on many new methods, among which is the identification through mRNA recovered from body fluid. Except mRNA profiling, microRNA and DNA methylation can also be used for body fluid identification.[14],[15],[16] Although mRNA profiling research has been carried out for years, it was seldom applied to real cases. Our results showed that this method could answer the urgent call of local police's demand and make sense in judiciary trial. Nevertheless, mRNA profiling is not as accurate as DNA analysis, which still needs more tissue-specific mRNA markers found to support the hypothesis. Furthermore, an inter-laboratory efficiency test protocol and guidelines are needed to make sure it is effective and can be widely used.

Singleplex versus multiplex

In contrast to miRNA, mRNA profiling can detect over 20 markers at a time.[8],[9],[17] It may save time and samples if we use multiplex PCR for identification. However, when it applies to routine cases, it seems to be more reliable to use singleplex PCR because of the unknown composition of the sample. In the routine testing of our laboratory, it is often difficult for a multiplex amplification system to obtain positive results for all markers, while a singleplex amplification system can achieve this. This may be related to the lower RNA content of some samples. Unless the volume of the sample is too low to detect enough markers, singleplex is better to explain what the sample contains. When the type of body fluid is unknown, a multiplex system can be considered for preliminary screening, and then a singleplex system can be used for confirmation. Since the sample of the case is affected by a variety of environmental factors, its RNA quantification will be lower than the minimum detection limit. Our laboratory also compared the amplification results with increasing PCR cycles, and the results showed that the amplification results at 36–40 cycles were reliable.

Result expression

Unlike STR profiling, it is more difficult to explain mRNA profiling results. First, cross-reactions are common, especially in body fluids that contain epithelial cells (saliva, vaginal fluids, and skin). Second, the threshold of relative fluorescence units (RFU) is difficult to establish, being influenced by the individual, body fluid type, and amplification conditions. It is necessary to perform parallel reactions to obtain reproducible results. Third, only one marker is not enough to identify a specific type of body fluid. Hence, a scoring system is proposed to evaluate the threshold. Roeder and Hass have developed a system to identify different body fluids, in which every type of body fluid will only resort to five markers to identify or exclude.[10] Fourth, the results of a singleplex amplification system and a multiplex amplification system should not be used in combination. Otherwise, the interpretation of the results may be biased. Fifth, to avoid false-positive results due to nonspecific amplification, it is necessary to repeat the amplification process. Finally, it is indispensable to let specialized investigators explain mRNA profiling results.[6]


  Conclusion Top


As a beneficial supplement of DNA analysis, mRNA profiling can offer additional information on the crime scene samples to help reconstruct the crime. Our results demonstrate that mRNA profiling can be applied to routine crime investigations as an aid. However, further validation works need to be done to prove its robustness and reliability.

Acknowledgment

We thank Kangyun Ai, Song Chen, Anquan Ji, Kaihui Liu, Yunying Ge, and Ying Zhang for their valuable advice for the case analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Tobe SS, Watson N, Daéid NN. Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA. J Forensic Sci 2007;52:102-9.  Back to cited text no. 1
    
2.
Fleming RI, Harbison S. The use of bacteria for the identification of vaginal secretions. Forensic Sci Int Genet 2010;4:311-5.  Back to cited text no. 2
    
3.
Juusola J, Ballantyne J. Messenger RNA profiling: A prototype method to supplant conventional methods for body fluid identification. Forensic Sci Int 2003;135:85-96.  Back to cited text no. 3
    
4.
Haas C, Hanson E, Anjos MJ, Ballantyne KN, Banemann R, Bhoelai B, et al. RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: Results of a fourth and fifth collaborative EDNAP exercise. Forensic Sci Int Genet 2014;8:203-12.  Back to cited text no. 4
    
5.
Jakubowska J, Maciejewska A, Pawłowski R. mRNA profiling for vaginal fluid and menstrual blood identification. Methods Mol Biol 2016;1420:33-42.  Back to cited text no. 5
    
6.
Juusola J, Ballantyne J. Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int 2005;152:1-12.  Back to cited text no. 6
    
7.
van den Berge M, Carracedo A, Gomes I, Graham EA, Haas C, Hjort B, et al. A collaborative European exercise on mRNA-based body fluid/skin typing and interpretation of DNA and RNA results. Forensic Sci Int Genet 2014;10:40-8.  Back to cited text no. 7
    
8.
Xu Y, Xie J, Cao Y, Zhou H, Ping Y, Chen L, et al. Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification. PLoS One 2014;9:e100123.  Back to cited text no. 8
    
9.
Roeder AD, Haas C. mRNA profiling using a minimum of five mRNA markers per body fluid and a novel scoring method for body fluid identification. Int J Legal Med 2013;127:707-21.  Back to cited text no. 9
    
10.
Fleming RI, Harbison S. The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids. Forensic Sci Int Genet 2010;4:244-56.  Back to cited text no. 10
    
11.
Hanson EK, Ballantyne J. Highly specific mRNA biomarkers for the identification of vaginal secretions in sexual assault investigations. Sci Justice 2013;53:14-22.  Back to cited text no. 11
    
12.
Lindenbergh A, Maaskant P, Sijen T. Implementation of RNA profiling in forensic casework. Forensic Sci Int Genet 2013;7:159-66.  Back to cited text no. 12
    
13.
An JH, Shin KJ, Yang WI, Lee HY. Body fluid identification in forensics. BMB Rep 2012;45:545-53.  Back to cited text no. 13
    
14.
Zubakov D, Boersma AW, Choi Y, van Kuijk PF, Wiemer EA, Kayser M. MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation. Int J Legal Med 2010;124:217-26.  Back to cited text no. 14
    
15.
Silva SS, Lopes C, Teixeira AL, Carneiro de Sousa MJ, Medeiros R. Forensic miRNA: Potential biomarker for body fluids? Forensic Sci Int Genet 2015;14:1-10.  Back to cited text no. 15
    
16.
An JH, Choi A, Shin KJ, Yang WI, Lee HY. DNA methylation-specific multiplex assays for body fluid identification. Int J Legal Med 2013;127:35-43.  Back to cited text no. 16
    
17.
Lindenbergh A, de Pagter M, Ramdayal G, Visser M, Zubakov D, Kayser M, et al. A multiplex (m) RNA-profiling system for the forensic identification of body fluids and contact traces. Forensic Sci Int Genet 2012;6:565-77.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

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