mRNA profiling using a minimum of five mRNA markers per body fluid and a novel scoring method for body fluid identification

International Journal of Legal Medicine - Tập 127 - Trang 707-721 - 2012
Amy D. Roeder1, Cordula Haas2
1Orchid Cellmark Ltd., Abingdon, UK
2Institute of Legal Medicine, University of Zurich, Zurich, Switzerland

Tóm tắt

Messenger RNA (mRNA) expression varies among cell types; therefore, analyses for the presence of particular mRNAs can be used to identify biological fluids in forensic samples. For this work, several novel markers were characterized for saliva, cervicovaginal fluid (CVF), blood, and menstrual blood (MB). The new markers were used in combination with previously described markers to develop four multiplex polymerase chain reaction assays. These multiplexes incorporate two housekeeping and a minimum of five markers for each of the following forensically relevant body fluids: semen, saliva, CVF, blood, and MB. A large number of samples (>200) were analyzed to determine specificity of each marker. The majority of the markers were detected at low frequencies in non-target body fluids. Because markers were not specific to their respective target body fluids, a scoring system was developed to minimize the chances of misidentification of a sample due to marker expression in a non-target body fluid. Each marker was given a numerical value related to its “correct” (target body fluid) versus “incorrect” (non-target body fluid) expression in samples of known origin. For each of the five body fluids, the marker values of those mRNA markers that were present in a sample were added to produce a body fluid score. Threshold scores were then determined for the identification of each body fluid. Although this study highlights the complexity of body fluid identification, particularly in differentiating blood and MB, the use of threshold scores allowed for reliable body fluid identification in the samples tested.

Tài liệu tham khảo

Virkler K, Lednev IK (2009) Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int 188:1–17 Baker DJ, Grimes EA, Hopwood AJ (2011) D-dimer assays for the identification of menstrual blood. Forensic Sci Int 212:210–214 Virkler K, Lednev IK (2008) Raman spectroscopy offers great potential for the nondestructive confirmatory identification of body fluids. Forensic Sci Int 181:e1–e5 French CEV, Jensen CG, Vintiner SK, Elliot DA, McGlashan SR (2008) A novel histological technique for distinguishing between epithelial cells in forensic casework. Forensic Sci Int 178:1–6 Sikirzhytskaya A, Sikirzhytski V, Lednev IK (2112) Raman spectroscopic signature of vaginal fluid and its potential application in forensic body fluid identification. Forensic Sci Int 216:44–48 Alvarez M, Juusola J, Ballantyne J (2004) An mRNA and DNA co-isolation method for forensic casework samples. Anal Biochem 335:289–298 Bauer M, Patzelt D (2002) Evaluation of mRNA markers for the identification of menstrual blood. J Forensic Sci 47:1278–1282 Bauer M, Patzelt D (2003) A method for simultaneous RNA and DNA isolation from dried blood and semen stains. Forensic Sci Int 136:76–78 Bowden A, Fleming R, Harbison SA (2011) A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ™ system. Forensic Sci Int Genet 5:64–68 Cossu C, Germann U, Kratzer A, Bär W, Haas C (2009) How specific are vaginal secretion mRNA-markers HBD1 and MUC4? Forensic Sci Int Genet Suppl Ser 2:536–537 Fang R, Manohar CF, Shulse C et al (2006) Real-time PCR assays for the detection of tissue and body fluid specific mRNAs. Int Congr Ser 1288:685–687 Fleming RI, Harbison SA (2010) The use of bacteria for the identification of vaginal secretions. Forensic Sci Int Genet 4:311–315 Fleming RI, Harbison SA (2010) The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids. Forensic Sci Int Genet 4:244–256 Haas C, Klesser B, Maake C, Bär W, Kratzer A (2009) mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. Forensic Sci Int Genet 3:80–88 Haas C, Muheim C, Kratzer A, Bär W, Maake C (2009) mRNA profiling for the identification of sperm and seminal plasma. Forensic Sci Int Genet Suppl Ser 2:534–535 Haas C, Hanson E, Bär W et al (2011) mRNA profiling for the identification of blood—results of a collaborative EDNAP exercise. Forensic Sci Int Genet 5:21–26 Haas C, Hanson E, Kratzer A, Bär W, Ballantyne J (2011) Selection of highly specific and sensitive mRNA biomarkers for the identification of blood. Forensic Sci Int Genet 5:449–458 Juusola J, Ballantyne J (2005) Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int 152:1–12 Noreault-Conti TL, Buel E (2007) The use of real-time PCR for forensic stain identification. Promega Profiles DNA 10:3–5 Nussbaumer C, Gharehbaghi-Schnell E, Korschineck I (2006) Messenger RNA profiling: a novel method for body fluid identification by real-time PCR. Forensic Sci Int 157:181–186 Patel G, Peel C (2008) Identifying the origin of cells. Forensic Sci Int Genet Suppl Ser 1:574–576 Richard MLL, Harper KA, Craig RL, Onorato AJ, Robertson JM, Donfack J (2012) Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis. Forensic Sci Int Genet 6:452–460 Setzer M, Juusola J, Ballantyne J (2008) Recovery and stability of RNA in vaginal swabs and blood, semen, and saliva stains. J Forensic Sci 53:296–305 Zubakov D, Hanekamp E, Kokshoorn M, van IJcken W, Kayser M (2008) Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples. Int J Legal Med 122:135–142 Zubakov D, Kokshoorn M, Kloosterman A, Kayser M (2009) New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains. Int J Legal Med 123:71–74 Noreault-Conti TL, Buel E (2008) Development of an RNA-based screening assay for forensic stain identification. 19th International Symposium on Human Identification. http://www.promega.com/~/media/files/resources/conference%20proceedings/ishi%2019/oral%20presentations/buel.ashx?la=en. Accessed 2 December 2011 Xie H, Rhodus NL, Griffin RJ, Carlis JV, Griffin TJ (2005) A catalogue of human saliva proteins identified by free flow electrophoresis-based peptide separation and tandem mass spectrometry. Mol Cell Proteomics 4:1826–1830 Hu S, Xie Y, Ramachandran P et al (2005) Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry. Proteomics 5:1714–1728 Hruz T, Laule O, Szabo G et al (2008) Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinformatics 2008:420747. doi:10.1155/2008/420747 Jain E, Bairoch A, Duvaud S et al (2009) Infrastructure for the life sciences: design and implementation of the UniProt website. BMC Bioinforma 10:136 Vásquez A, Jakobsson T, Ahrné S, Forsum U, Molin G (2002) Vaginal Lactobacillus flora of healthy Swedish women. J Clin Microbiol 40:2746–2749 Pavlova SI, Kilic AO, Kilic SS, So JS, Nader-Macias ME, Simoes JA, Tao L (2002) Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences. J Appl Microbiol 92:451–459 Carson DD, Lagow E, Thathiah A et al (2002) Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Mol Hum Reprod 8:871–879 Mahoney DJ, Safdar A, Parise G (2008) Gene expression profiling in human skeletal muscle during recovery from eccentric exercise. Am J Physiol Regul Integr Comp Physiol 294:R1901–R1910 Raj A, van Oudenaarden A (2008) Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135:216–226 Su AI, Wiltshire T, Batalov S et al (2004) A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci USA 101:6062–6067 Whitehead A, Crawford DL (2005) Variation in tissue-specific gene expression among natural populations. Genome Biol 6:R13 Helo P, Cronin AM, Danila DC et al (2009) Circulating prostate tumor cells detected by reverse transcription-PCR in men with localized or castration-refractory prostate cancer: concordance with cell search assay and association with bone metastases and with survival. Clin Chem 55:765–773 Moreno JG, Croce CM, Rischer R et al (1992) Detection of hematogenous micrometastasis in patients with prostate cancer. Cancer Res 52:6110–6112 Hochmeister MN, Budowle B, Rudin O et al (1999) Evaluation of prostate-specific antigen (PSA) membrane test assays for the forensic identification of seminal fluid. J Forensic Sci 44:1057–1060 Pang BCM, Cheung BKK (2007) Identification of human semenogelin in membrane strip test as an alternative method for the detection of semen. Forensic Sci Int 169:27–31 Romero-Montoya L, Martínez-Rodríguez H, Pérez MA, Argüello-García R (2011) Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases. Forensic Sci Int 206:111–118 Benschop CCG, Wiebosch DC, Kloosterman AD, Sijen T (2010) Post-coital vaginal sampling with nylon flocked swabs improves DNA typing. Forensic Sci Int Genet 4:115–121 Dziak R, Parker L, Collins V, Johnston S (2011) Providing evidence based opinions on time since intercourse (TSI) based on body fluid testing results of internal samples. Can Soc Forensic Sci J 44:59–69 Casey DG, Price J (2010) The sensitivity and specificity of the RSID™-saliva kit for the detection of human salivary amylase in the forensic science laboratory, Dublin, Ireland. Forensic Sci Int 194:67–71 Hedman J, Dalin E, Rasmusson B, Ansell R (2011) Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs. Forensic Sci Int Genet 5:194–198 Pang BCM, Cheung BKK (2008) Applicability of two commercially available kits for forensic identification of saliva stains. J Forensic Sci 53:1117–1122 Gipson IK, Spurr-Michaud S, Moccia R et al (1999) MUC4 and MUC5B transcripts are the prevalent mucin messenger ribonucleic acids of the human endocervix. Biol Repro 60:58–64 Fleming DC, King AE, Williams ARW, Critchley HOD, Kelly RW (2003) Hormonal contraception can suppress natural antimicrobial gene transcription in human endometrium. Fertil Steril 79:856–863 Eschenbach DA, Davick PR, Williams BL et al (1989) Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 27:251–256 Gupta K, Stapleton AE, Hooton TM, Roberts PL, Fennell CL, Stamm WE (1998) Inverse association of H2O2-producing lactobacilli and vaginal Escherichia coli colonization in women with recurrent urinary tract infections. J Infect Dis 178:446–450 Keane FEA, Ison CA, Taylor-Robinson D (1997) A longitudinal study of the vaginal flora over a menstrual cycle. Int J STD AIDS 8:489–494 Hammerschlag MR, Alpert S, Rosner I et al (1978) Microbiology of the vagina in children: normal and potentially pathogenic organisms. Pediatrics 62:57–62 Cauci S, Driussi S, De Santo D et al (2002) Prevalence of bacterial vaginosis and vaginal flora changes in peri- and postmenopausal women. J Clin Microbiol 40:2147–2152 Pabich WL, Fihn SD, Stamm WE, Scholes D, Boyko EJ, Gupta K (2003) Prevalence and determinants of vaginal flora alterations in postmenopausal women. J Infect Dis 188:1054–1058 Cox M (1991) A study of the sensitivity and specificity of four presumptive tests for blood. J Forensic Sci 36:1503–1511 Johnston S, Newman J, Frappier R (2003) Validation study of the Abacus diagnostics ABAcard® Hematrace® membrane test for the forensic identification of human blood. Can Soc Forens Sci J 36:173–183 Tobe SS, Watson N, Daéid NN (2007) Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA. J Forensic Sci 52:102–109 Fraser IS, McCarron G, Markham R, Resta T (1985) Blood and total fluid content of menstrual discharge. Obstet Gynecol 65:194–198 Fraser IS, Warner P, Marantos PA (2001) Estimating menstrual blood loss in women with normal and excessive menstrual fluid volume. Obstet Gynecol 98:806–814 Rybo G (1966) Clinical and experimental studies on menstrual blood loss. Acta Obstet Genecol Scand 45(Suppl 7):1–23 Steger K, Pauls K, Klonisch T, Franke FE, Bergmann M (2000) Expression of protamine-1 and -2 mRNA during human spermiogenesis. Mol Hum Reprod 6:219–225