Messenger RNA Profiling for Forensic Body Fluid Identification:Research and Applications

WANG Zheng,ZHANG Su-hua,ZHOU Di,ZHAO Shu-min,LI Cheng-tao

（1.Shanghai Key Laboratory of Forensic Medicine,Institute of Forensic Science,Ministry of Justice,P.R.China,Shanghai 200063,China;2.Department of Forensic Genetics,West China School of Basic Science and Forensic Medicine,Sichuan University,Chengdu 610041,China）

Messenger RNA Profiling for Forensic Body Fluid Identification:Research and Applications

WANG Zheng1,2,ZHANG Su-hua1,ZHOU Di2,ZHAO Shu-min1,LI Cheng-tao1

（1.Shanghai Key Laboratory of Forensic Medicine,Institute of Forensic Science,Ministry of Justice,P.R.China,Shanghai 200063,China;2.Department of Forensic Genetics,West China School of Basic Science and Forensic Medicine,Sichuan University,Chengdu 610041,China）

Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link between sample donors and actual criminal acts.However,the conventional body fluid identification methods are prone to various limitations,such as time consumption,intensive labor,nonparallel manner,varying degrees of sensitivity and limited specificity.Recently,the analysis of cell-specific messenger RNA expression（mRNA profiling）has been proposed to supplant conventional methods for body fluid identification.Since 2011,the collaborative exercises have been organized by the European DNA Profiling Group（EDNAP）in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification.The major advantages of mRNA profiling,compared to the conventional methods,include higher sensitivity,greater specificity,the ability of detecting several body fluids in one multiplex reaction,and compatibility with current DNA extraction and analysis procedure.In the current review,we provided an overview of the present knowledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possible practical application to forensic casework.

forensic genetics;mRNA profiling;review[publication type];body fluid identification

Article IC:1004-5619（2013）05-0368-07

Introduction

The routine practice in forensic casework analysis typically includes a preliminary screening of evidentiary items taken from crime scenes to identify the possible tissue or body fluid origin of biological material.The presence of biological material such as saliva,blood and semen stains can indicate the location of potential sources of DNA,which can be used to identify the donor of the biological material through DNA typing technology.The conventional serology-, biochemistry- and immunology-based methods for body fluid identification are costly in terms of both labor and time required for operation and the amount of sample consumed during the performance of each assay[1-7].The deficiencies of the conventional methods and the modern DNA testing technology obtaining the DNA profile from only a few nucleated cells or touch samples have resulted in a trend to bypass body fluid identification and process DNA analysis directly[8].However,in forensic casework,especially in the cases of sexual assault and child sexual abuse,to uncover the criminal nature requires not only identification of the DNA profile,but also confirmation of DNA origin.In a scenario of a sexual assault where the victim’s DNA profile is recovered from blood from the suspect’s clothing,the defense argues that the blood comes from the victim’s nose or mouth when she is punched by the suspect.The ability to identify blood as menstrual in origin,as opposed to peripheral blood,would more strongly support the allegation of a sexual assault.Another example is child sexual abuse where the stepfather’s DNA is found in the child’s underwear and bedding,and the stepfather claims the presence due to casual and frequent contact.However,the finding that DNA originated from a semen stain would more strongly support the allegation of a sexual assault.

In cells, mRNA molecules transcribed from DNA are responsible for the transfer of genetic information to ribosomes,determining the amino acid sequence of protein synthesis.Different body fluids are made up of multiple types of cells,and each cell type has a unique pattern of gene expression known as the transcriptome[9].Forensic geneticists will be mostly interested in the multicellular transcriptome concept,which refers to the collection of different gene expression in a particular body fluid.

RNA is notorious for its rapid postmortem and in vitro decay because of the ubiquitously present ribonucleases[10-11].However,the developments of RNA technology together with reports of unexpectedly high stability in certain conditions have stimulated forensic scientists to start exploring the RNA world[12-17]. Setzer et al.[14]exposed body fluid stains to a rangeof environmental conditions and demonstrated that RNA was detectable in some stains stored at room temperature even after 547 days.Using whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples,Zubakov et al.[15]identified 14 mRNA markers that showed stable expression patterns in stains after up to 180 days of storage,and some of these markers showed reliable amplification in 16 year-old blood stains[16]. These reports demonstrated that RNA was stable in biological stains and could be recovered in sufficient quantity and quality for analysis.

Because the quantity or quality of biomaterial is often limited in a real case,there are two prerequisites to mRNA profiling as a routine procedure, one the ability to co-extract nuclear DNA and mRNA from the same sample without compromising the potential DNA profile,and another the detection method which is compatible with current DNA analysis procedure.Much work has been done to address the challenge of mRNA and DNA co-isolation,as well as the validation of methods to analyze certain transcripts and suitable sets of stable mRNA markers[18-22]. The number of forensic RNA papers has risen steadily and a number of mRNA markers and housekeeping genes have been used to identify the forensic relevant body fluids:venous blood,saliva,semen, vaginal secretions and menstrual blood[23-44]（Table 1 and 2）.The recent procedure for mRNA-based body fluid identification involved RNA extraction using the DNA/RNA extraction kit,followed by multiplex PCR of specific mRNAs markers with fluorescence-labeled primers,and capillary electrophoresis（CE）of amplification products and automatic signal detection[40-43].The current review provided an overview of the most recent developments,outlining the procedures and considerations of mRNA profiling for forensic body fluid identification.

Table 1Reported mRNA markers for forensic body fluid identification

Table 2Reported housekeeping genes for forensic body fluid identification

mRNA profiling in forensic body fluid identification

Different types of the cells in body fluids contain different collections of mRNAs that are unique to the cell type.Therefore,mRNA profiling offers an opportunity to develop body fluid-specific assays which could identify the specifically body fluid by unique mRNAs,such as venous blood versus semen.After crucial methodological progress was made in RNA extraction,reverse transcription PCR （RT-PCR）[45]and cDNA quantification,i.e.the invention of quantitative PCR（qPCR）[46],mRNA expression analysis for the identification of body fluids analysis was explored.

Using RT-PCR and agarose gel electrophoresis, Bauer et al.[23]were first to explore the forensic potential of mRNA for the identification of body fluids,and reported that MMP and protamine mRNA could be used to identify menstrual blood and spermatozoa,respectively[24-25].Juusola et al.[26]proved RNA in biological stains could be recovered in sufficient quantity and quality for analysis using three reference genes,ACTB,GAPDH and S15,and proposed STATH,HTN3,PRB1,PRB2 and PRB3 for the positive identification of saliva.In their updated experiment,Juusola et al.[27]demonstrated a multiplex end-point PCR method for definitive identification of blood,saliva,semen,and vaginal secretions.Using eight body fluid-specific mRNAs,i.e.,SPTBand PBGD for blood,STATH and HTN3 for saliva, PRM1 and PRM2 for semen,and hBD-1 and MUC4 for vaginal secretions,the octaplex assay could detect each of the four body fluids as single or mixed stains.Since the successful identification of body fluids was possible with 200pg-12 ng of input RNA, the sensitivity of this multiplex assay was suitable for forensic casework.

Many mRNAs are not completely body fluid-specific,present or absent,but usually show high expression levels in target body fluid and very low ones in the others.Thus,a qualitative analysis,e.g. end-point PCR,might not be appropriate,but rather quantitative analyses are then suitable.In 2006, quantitative approach（qPCR）was developed by Nussbaumer et al.[28]to improve the resolution and enhance sensitivity,showing that KLK was only detectable in semen but not in other body fluids.In this context,mRNA analysis via end-point PCR is sufficient.They also showed that transcripts of HBA and MUC were highly abundant in blood and vaginal secretion,respectively,but also detectable in other body fluids.This condition requires a quantitative method.In qPCR analysis,proper normalization strategy played a central role in minimizing potential variation that could mask or exaggerate biologically meaningful changes[47-49].Without normalization,analyses might lead to false positive or false negative results.In 2007,Juusola et al.[29]introduced housekeeping gene GAPDH as the normalization of the expression of body fluid-specific mRNAs.They established four triplex assays,each including two body fluid-specific mRNAs and GAPDH,for the identification of blood（ALAS2 and SPTB）,saliva （STATH and HTN3）,semen（PRM1 and PRM2）, and menstrual blood（MMP7 and MMP10）,respectively.

In 2009,Haas et al.[31]reported a multiplex RT-PCR and a multiplex qPCR using previously reported mRNA markers,and demonstrated that both end-point PCR and qPCR were suitable for the identification of body fluids in forensic stains.The ability of qPCR to quantitate target molecules is important in searching the mRNA with large-magnitude fold-change between body fluids or only expression in a particular body fluid.When specific markers had been identified by qPCR,however,incorporating mRNA profiling strategies into current DNA analysis pipelines would be preferable.Haas et al.[35]evaluated eight reported blood-specific mRNA markers,HBB,HBA,ALAS2,CD3G,ANK1,PBGD, SPTB and AQP9 for their sensitivities,specificities and performance with casework samples.They observed the varying levels of expression on CE and all the markers（except AQP9）relatively sensitive requiring as little as 1ng of RNA input.Lindenbergh et al.[37]conducted a more comprehensive study in 2012,developing an end-point PCR assay that simultaneously amplified 19 markers.This mul-tiplex assay analyzed three reference genes（GAPDH, 18S-rRNA and ACTB）,three for blood（HBB, CD93 and AMICA1）,two for semen（SEMG1 and PRM1）,two for saliva（HTN3 and STATH）,two for menstrual secretion（MMP7 and MMP11）,two for vaginal mucosa（hBD-1 and MUC4）,three for general mucosa（KRT4,SPRR2A and KRT13）and two for skin（CDSN and LOR）.The assay on CE possessed high sensitivity as full RNA profiles were obtained using 0.05 μL body fluid starting material, whereas full DNA profiles were obtained with 0.1 μL.

Recently,EDNAP organized three collaborative exercises on mRNA profiling for the identification of body fluids[40-42].For the first time,16 laboratories, most with no prior experience with RNA,were invited to evaluate the robustness and reproducibility of mRNA profiling for blood identification using the 3 blood-specific markers,HBB,SPTB and PBGD, the results demonstrating that all but one of the 16 participating laboratories managed to isolate and analyze blood specific-mRNA from the provided stains. For the second,7 blood-specific markers divided into two multiplex systems,namely a“high sensitivity”duplex（HBA,HBB）and a“moderate sensitivity”pentaplex（ALAS2,CD3G,ANK1,PBGD and SPTB）were critically valuated with bona fide or mock casework samples,even with old and compromised stains.A third collaborative exercise was organized in order to test forensically suitable saliva （HTN3,STATH and MUC7）and semen markers （PSA,PRM2,SEMG1,TGM4 and PRM1）.The results of these collaborative exercises supported the potential use of an mRNA-based system for the identification of body fluids in forensic casework that was compatible with current DNA analyses.

In the current review,we introduced the mRNA analysis procedure（Fig.1）recommended by EDNAP.

Fig.1Proposed schema for the analysis of biological stains using mRNA profiling

The workflow of mRNA for identification of body fluids

RNA extraction and quantification

Chemical extraction（e.g.TRIzol®Reagent,Invitrogen）and solid-phase extraction（e.g.EZ1 RNA Universal Tissue Kit,Qiagen）are two classical methods to prepare RNA,which can provide pure RNA. However,simultaneous extraction of mRNA and DNA from the same fluid stain has to be considered for the limited amount of evidentiary sample.Performing an mRNA analysis on the RNA extracts will yield information regarding the identity of the stain, and the DNA analysis will reveal the donor’s identity.Currently,several optimized methods have been developed for simultaneously isolating mRNAand DNA from same stains[18-22].The most frequently used commercial co-extraction kit is Allprep DNA/RNA Mini Kit（Qiagen）,which allows the parallel processing of multiple samples in less than 40 minutes.Lysate is first passed through an AllPrep DNA spin column to selectively isolate DNA,and then through an RNeasy spin column to selectively isolate RNA.In order to avoid primer and nucleotide titration resulting from contamination of DNA, DNase treatment of RNA extracts has been recommended[40-43].It was observed that the TURBO DNA-freeTMKit（Ambion）removed contaminating DNA more efficiently than the on-column RNase-Free DNase Set（Qiagen）[43].

It is beneficial to put a defined amount（around 1-10 ng）of RNA into the RT reaction so as to avoid false positive and negative signals.EDNAP recommends the following RNA quantitative methods:Quant-iTTMRNA Assay Kit（Invitrogen）with the Qubit®fluorometer;Quant-iTTMRiboGreen® RNAAssay Kit（Invitrogen）with a fluorescence microplate reader（low and high range protocol option）or Bioanalyzer（Agilent）.

Reverse transcription and end-point PCR

For the reverse transcription（RT）reaction,several kits are available,whose suitability for use with forensic biological samples has been demonstrated by EDNAP[42].Zubakov et al.[16]tested random hexamer primers,oligo（dT）20primers,and gene specific primers for sensitivity effects of the reverse transcription technique.All three kinds of primers allowed successful amplification on a similar level of sensitivity,but experimental reproducibility was slightly better for cDNA produced by random hexamer primers than for gene-specific or oligo（dT）20primers.Thus,SuperScript®ⅢReverse Transcriptase（Invitrogen）with random hexamer primers is the most commonly used in forensic RNA research. The RT minus control,with no reverse transcriptase added,should be included to detect possible DNA contamination.

An important advantage of mRNA profiling is the ability to use multiplex markers for identifying the multiple body fluids simultaneously.However, the development of a robust multiplex PCR system can be a challenge,in that not all products are amplified with equal efficiency and nonspecific noises are often observed.Qiagen®Multiplex PCR Kit,the first commercially available kit for multiplex PCR, contains a master mix whose composition and elements were specifically developed,and minimizes the need for optimization（e.g.split peaks,noisy baseline）.Primers should be designed to overlap exon-exon-junctions or span an intron in order to ensure that the obtained products are not due to the presence of contaminating DNA.Meanwhile, the amplification blank（no template added）should be included to detect possible contamination during PCR（e.g.contamination of the master mix with template）and during CE（e.g.re-using sample septa or buffer septa,crosstalk or carryover）,while a positive control marker which expresses in all tissue types would confirm a successful analysis.

Post-PCR purification and CE

Post PCR purification,（e.g.MinElute PCR Purification Kit,Qiagen）is not necessary but recommendable,particularly for low template samples and multiplex assays.The use of post-PCR purification can increase peak heights from low-level samples, and eliminate primer peaks and dye blobs.For the multiplex assay,it can critically improve the quality of the multiplex results such as reduced appearance of split peaks,dye blobs,etc.

The threshold for a positive result is generally set to 100 RFUs（relative fluorescence units）,which is higher than that for STR typing to avoid inclusion of false positive result mainly due to the presence of dye blobs and baseline noise.

Further work

While the current forensic DNA profiling based on STRs allows identification of biological sample donors,recent advances in forensics have suggested using other kinds of markers in order to add more informative layers to the evidence.Various types of markers（e.g.mRNA,microRNA[50-55]and methylation[56-60]）have been proposed for forensic body fluid identification.Among these markers,mRNAs have been most rigorously investigated,the number of specific markers sufficient for the identification of forensically relevant body fluids.Undoubtedly,mRNA profiling has vast potential for the identification of body fluid.However,important issues need to be addressed while performing RNA profiling in forensic casework.

Firstly,RNA quantitation prior to the reverse transcription reaction can eliminate some of the adverse affects of adding too little input RNA（products below the analytical detection limits）or too much input RNA（saturation,extraneous peaks and cross-reactivity）.However,it is not suitable using NanoDrop Spectrophotometer and the Agilent Bioanalyzer for assessing RNA quantity and quality in forensic samples[43].The quantity of the extracted RNA from forensic stains is usually below the detection limit of the NanoDrop spectrophotometer and the RNA quality is often poorly assessed with the Agilent Bioanalyzer（according to RIN metric）.Additionally,fluorescence based RNA quantification methods suggested by EDNAP are also not human-specific.Thus,the presence of bacterial RNA, which is found in abundance in vaginal swabs or saliva,will affect the determination of the amount of total RNA present in body fluids.A sensitive and robust human specific RNA quantification system will be developed to give accurate quantification of human total RNA in forensic stains.

Next,the most impending pitfall of mRNA profiling is the problem of“specificity”.The“specificity”in the concept of mRNA profiling indicates that expression of a particular mRNA either is only observed in one body fluid or observed in target body fluid among various body fluids.Since some cross-reactivity reported,it presented a greater challenge in terms of interpretation.More refined analysis interpretation metrics and proper interpretation guidelines should be developed to provide a robust objective statistical approach for declaring a sample to be negative or positive with respect to the presence of a particular body fluid.

In addition,it is imperative that precautions be taken to create an RNase free environment with RNA in the laboratory.Such precautions include a separate RNA working place,a clean working area with RNase destroying solution,RNase free plastic ware,separate pipettes and regular changing of gloves.John M.Butler,world-famous forensic geneticist,noted in his 2011 book Advanced Topics in Forensic DNA Typing:Methodology,“If the samples are not handled properly in the initial stages of an investigation,then no amount of hard work in the final analytical or data interpretation steps can compensate.”

Acknowledgements

This review was supported by the grants from the National Key Technology Researchamp;Development Program of the Ministry of Science and Technology of People’s Republic of China（2012BAK16B01）and the National Natural Science Foundation of People’s Republic of China（No.81222041,81172908）.

[1]Khaldi N, Miras A, Botti K,et al. Evaluation of three rapid detection methods for the forensic identification of seminal fluid in rape cases[J]. J Forensic Sci, 2004, 49（4）:749-753.

[2]Tobe SS, Watson N, Daéid NN.Evaluation of six presumptive tests for blood,their specificity,sensitiv-ity, and effect on high molecular-weight DNA[J]. J Forensic Sci, 2007, 52（1）:102-109.

[3]Myers JR, Adkins WK. Comparison of modern techniques for saliva screening[J]. J Forensic Sci, 2008, 53（4）:862-867.

[4]Virkler K, Lednev IK. Analysis of body fluids for forensic purposes:from laboratory testing to non-destructive rapid confirmatory identification at a crime scene[J]. Forensic Sci Int, 2009, 188（1-3）:1-17.

[5]An JH,Shin KJ, Yang WI, et al. Body fluid identification in forensics[J]. BMB Rep, 2012,45（10）:545-553.

[6]Bauer M. RNA in forensic science[J]. Forensic Sci Int Genet, 2007, 1（1）:69-74.

[7]Vennemann M, Koppelkamm A. mRNA profiling in forensic genetics Ⅰ: Possibilities and limitations[J]. Forensic Sci Int, 2010, 203（1-3）:71-75.

[8]Fourney RM, DesRoches AN, Buckle JL, et al. Recent progress in processing biological evidence and forensic DNA profiling:A review 2004 to 2007[C]. In Interpol 15th international forensic science symposium, 2007:635-719.

[9]Caron H, van Schaik B, van der Mee M, et al. The human transcriptome map:clustering of highly expressed genes in chromosomal domains[J]. Science, 2001, 291（5507）:1289-1292.

[10]van Hoof A, Parker R.Messenger RNA degradation: beginning at the end[J]. Curr Biol, 2002, 12（8）:R285-R287.

[11]Lee J, Hever A, Willhite D, et al. Effects of RNA degradation on gene expression analysis of human postmortem tissues[J]. FASEB J, 2005, 19（10）:1356-1358.

[12]Karlsson H, Guthenberg C, von Döbeln U, et al. Extraction of RNA from dried blood on filter papers after long-term storage[J]. Clin Chem, 2003, 49（6 Pt 1）:979-981.

[13]Ferri G, Bini C, Ceccardi S, et al. Successful identification of two years old menstrual bloodstain by using MMP-11 shorter amplicons[J]. J Forensic Sci, 2004, 49（6）:1387.

[14]Setzer M, Juusola J, Ballantyne J. Recovery and stability of RNA in vaginal swabs and blood,semen, and saliva stains[J]. J Forensic Sci, 2008, 53（2）:296-305.

[15]Zubakov D, Hanekamp E, Kokshoorn M, et al. Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples[J]. Int J Legal Med, 2008, 122（2）:135-142.

[16]Zubakov D, Kokshoorn M, Kloosterman A, et al. New markers for old stains:stable mRNA markers for blood and saliva identification from up to 16-yearold stains[J]. Int J Legal Med, 2009, 123（1）:71-74.

[17]Kohlmeier F, Schneider PM. Successful mRNA profiling of 23 years old blood stains[J]. Forensic Sci Int Genet, 2012, 6（2）:274-276.

[18]Bauer M, Patzelt D. A method for simultaneous RNA and DNA isolation from dried blood and semen stains[J]. Forensic Sci Int, 2003, 136（1-3）:76-78.

[19]Alvarez M, Juusola J, Ballantyne J. An mRNA and DNA co-isolation method for forensic casework samples[J]. Anal Biochem, 2004, 335（2）:289-298.

[20]Bowden A, Fleming R, Harbison S.A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQTMsystem[J]. Forensic Sci Int Genet, 2011, 5（1）:64-68.

[21]Triant DA, Whitehead A. Simultaneous extraction of high-quality RNA and DNA from small tissue samples[J]. J Hered, 2009, 100（2）:246-250.

[22]Parker C, Hanson E, Ballantyne J. Optimization of dried stain co-extraction methods for efficient recovery of high quality DNA and RNA for forensic analysis[J]. Forensic Science International:Genetics Supplement Series, 2011, 3（1）:e309-e310.

[23]Bauer M, Kraus A, Patzelt D. Detection of epithelial cells in dried blood stains by reverse transcriptase-polymerase chain reaction[J]. J Forensic Sci, 1999, 44（6）: 1232-1236.

[24]Bauer M, Patzelt D.Evaluation of mRNA markers for the identification of menstrual blood[J]. J Forensic Sci, 2002, 47（6）:1278-1282.

[25]Bauer M, Patzelt D. Protamine mRNA as molecular marker for spermatozoa in semen stains[J]. Int J Legal Med, 2003, 117（3）:175-179.

[26]Juusola J, Ballantyne J. Messenger RNA profiling:a prototype method to supplant conventional methods for body fluid identification[J]. Forensic Sci Int, 2003, 135（2）:85-96.

[27]Juusola J, Ballantyne J. Multiplex mRNA profiling for the identification of body fluids[J]. Forensic Sci Int, 2005, 152（1）:1-12.

[28]Nussbaumer C, Gharehbaghi-Schnell E, Korschineck I. Messenger RNA profiling:a novel method for body fluid identification by real-time PCR[J]. Forensic Sci Int, 2006, 157（2-3）:181-186.

[29]Juusola J, Ballantyne J. mRNA profiling for body fluid identification by multiplex quantitative RT-PCR[J]. J Forensic Sci, 2007, 52（6）:1252-1262.

[30]Bauer M, Patzelt D. Identification of menstrual blood by real time RT-PCR:technical improvements and the practical value of negative test results[J]. Forensic Sci Int, 2008, 174（1）:55-59.

[31]Haas C, Klesser B, Maake C, et al. mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR[J]. Forensic Sci Int Genet, 2009, 3（2）:80-88.

[32]Haas C, Muheim C, Kratzer A, et al. mRNA profiling for the identification of sperm and seminal plasma[J]. Forensic Science International:Genetics Supplement Series, 2009, 2（1）:534-535.

[33]Sakurada K, Ikegaya H, Fukushima H,et al. Evalua-tion of mRNA-based approach for identification of saliva and semen[J]. Leg Med（Tokyo）, 2009, 11（3）: 125-128.

[34]Fleming RI, Harbison S. The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids[J]. Forensic Sci Int Genet, 2010, 4（4）:244-256.

[35]Haas C, Hanson E, Kratzer A, et al. Selection of highly specific and sensitive mRNA biomarkers for the identification of blood[J]. Forensic Sci Int Genet, 2011, 5（5）:449-458.

[36]Richard ML, Harper KA, Craig RL, et al. Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis[J]. Forensic Sci Int Genet, 2012, 6（4）:452-460.

[37]Lindenbergh A, de Pagter M, Ramdayal G, et al. A multiplex（m）RNA-profiling system for the forensic identification of body fluids and contact traces[J]. Forensic Sci Int Genet, 2012, 6（5）:565-577.

[38]Lindenbergh A, Maaskant P, Sijen T. Implementation of RNA profiling in forensic casework[J]. Forensic Sci Int Genet, 2013, 7（1）:159-166.

[39]Park SM, Park SY, Kim JH, et al. Genome-wide mRNA profiling and multiplex quantitative RT-PCR for forensic body fluid identification[J]. Forensic Sci Int Genet, 2013, 7（1）:143-150.

[40]Haas C, Hanson E, Bär W, et al. mRNA profiling for the identification of blood--results of a collaborative EDNAP exercise[J]. Forensic Sci Int Genet, 2011, 5（1）:21-26.

[41]Haas C, Hanson E, Anjos MJ, et al. RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise[J]. Forensic SciInt Genet, 2012, 6（1）:70-80.

[42]Haas C, Hanson E, Anjos MJ, et al. RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise[J]. Forensic Sci Int Genet, 2013,7（2）:230-239.

[43]Alonso A. DNA electrophoresis protocols for forensic genetics[M]. New York:Humana Press Inc., 2012:169-183.

[44]Wang YX, Zhu XJ, Jiao ZP, et al. A new method of identifying the peripheral blood and the menstrual blood[J]. Fa Yi Xue Za Zhi, 2012, 28（5）:359-361.

[45]Phang TW, Shi CY, Chia JN, et al. Amplification of cDNA via RT-PCR using RNA extracted from postmortem tissues[J]. J Forensic Sci, 1994, 39（5）:1275-1279.

[46]Nolan T, Hands RE, Bustin SA.Quantification of mRNA using real-time RT-PCR[J]. Nat Protoc, 2006, 1（3）:1559-1582.

[47]Wong ML, Medrano JF. Real-time PCR for mRNA quantitation[J]. Biotechniques, 2005, 39（1）:75-85.

[48]Huggett J, Dheda K, Bustin S, et al. Real-time RT-PCR normalisation;strategies and considerations[J]. Genes Immun, 2005, 6（4）:279-284.

[49]Bustin SA, Benes V, Nolan T, et al. Quantitative real-time RT-PCR--a perspective[J]. J Mol Endocrinol, 2005, 34（3）:597-601.

[50]Hanson EK, Lubenow H, Ballantyne J. Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs[J]. Anal Biochem, 2009, 387（2）:303-314.

[51]Zubakov D, Boersma AW, Choi Y, et al. MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation[J]. Int J Legal Med, 2010, 124（3）:217-226.

[52]Courts C, Madea B. Specific micro-RNA signatures for the detection of saliva and blood in forensic body-fluid identification[J]. J Forensic Sci, 2011, 56（6）: 1464-1470.

[53]Wang Z, Luo H, Pan X, et al. A model for data analysis of microRNA expression in forensic body fluid identification[J]. Forensic Sci Int Genet, 2012, 6（3）: 419-423.

[54]Wang Z, Zhang J, Luo H, et al. Screening and confirmation of microRNA markers for forensic body fluid identification[J]. Forensic Sci Int Genet, 2013, 7（1）:116-123.

[55]van der Meer D, Uchimoto ML, Williams G. Simultaneous analysis of micro-RNA and DNA for determining the body fluid origin of DNA profiles[J]. J Forensic Sci, 2013, 58（4）:967-971.

[56]Frumkin D, Wasserstrom A, Budowle B, et al. DNA methylation-based forensic tissue identification[J]. Forensic Sci Int Genet, 2011, 5（5）:517-524.

[57]Lee HY, Park MJ, Choi A, et al. Potential forensic application of DNA methylation profiling to body fluid identification[J]. Int J Legal Med, 2012, 126（1）: 55-62.

[58]An JH, Choi A, Shin KJ, et al. DNA methylation-specific multiplex assays for body fluid identification[J]. Int J Legal Med, 2013, 127（1）:35-43.

[59]Madi T, Balamurugan K, Bombardi R, et al. The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing[J]. Electrophoresis, 2012, 33（12）: 1736-1745.

[60]Wasserstrom A, Frumkin D, Davidson A,et al. Demonstration of DSI-semen--A novel DNA methylation-based forensic semen identification assay[J]. Forensic Sci Int Genet, 2013, 7（1）:136-142.

（Received date:2013-08-23）

（Editor:LIU Yan）

DF795.2

A

10.3969/j.issn.1004-5619.2013.05.013

Author:WANG Zheng（1984—）,Ph.D in forensic genetics; E-mail:wangzhengtim@163.com

LI Cheng-tao,Ph.D,research fellow, postgraduate tutor in forensic genetics;E-mail:lichengtaohla@ 163.com