Highlights
- •The body fluid specific markers correctly amplified in their corresponding body fluid.
- •The equalization of cDNA quantities reduced body fluid cross-reactivities.
- •Differences in the performance of assays were observed.
- •At least two body fluid specific markers must be detected for accurate interpretation of cDNA profiling.
- •The assays are not ready for immediate application in forensic casework.
Abstract
Several mRNA markers have been exhaustively evaluated for the identification of human
venous blood, saliva, and semen in forensic genetics. As new candidate human body
fluid specific markers are discovered, evaluated, and reported in the scientific literature,
there is an increasing trend toward determining the ideal markers for cDNA profiling
of body fluids of forensic interest. However, it has not been determined which molecular
genetics-based technique(s) should be utilized to assess the performance of these
markers. In recent years, only a few confirmatory, mRNA/cDNA-based methods have been
evaluated for applications in body fluid identification. The most frequently described
methods tested to date include quantitative polymerase chain reaction (qPCR) and capillary
electrophoresis (CE). However these methods, in particular qPCR, often favor narrow
multiplex PCR due to the availability of a limited number of fluorescent dyes/tags.
In an attempt to address this technological constraint, this study explored matrix-assisted
laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for human
body fluid identification via cDNA profiling of venous blood, saliva, and semen. Using
cDNA samples at 20 pg input phosphoglycerate kinase 1 (PGK1) amounts, body fluid specific markers for
the candidate genes were amplified in their corresponding body fluid (i.e., venous
blood, saliva, or semen) and absent in the remaining two (100% specificity). The results
of this study provide an initial indication that MALDI-TOF MS is a potential fluorescent
dye-free alternative method for body fluid identification in forensic casework. However,
the inherent issues of low amounts of mRNA, and the damage caused to mRNA by environmental
exposures, extraction processes, and storage conditions are important factors that
significantly hinder the implementation of cDNA profiling into forensic casework.
Keywords
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References
- Molecular Biology of the Cell.third ed. Garland Publishing, Inc., New York1994
- The human transcriptome map: clustering of highly expressed genes in chromosomal domains.Science. 2001; 291: 1289-1292
- Body fluid identification in forensics.BMB Rep. 2012; 45: 545-553
- mRNA profiling in forensic genetics I: possibilities and limitations.Forensic Sci. Int. 2010; 203: 71-75
- RNA in forensic science.Forensic Sci. Int. Genet. 2007; 1: 69-74
- mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR.Forensic Sci. Int. Genet. 2009; 3: 80-88
- mRNA profiling for the identification of sperm and seminal plasma.Forensic Sci. Int.: Genet. Suppl. Ser. 2009; 2: 534-535
- RNA/DNA co-analysis from blood stains – results of a second collaborative EDNAP exercise.Forensic Sci. Int. Genet. 2012; 6: 70-80
- Capillary electrophoresis of a multiplex reverse transcription-polymerase chain reaction to target messenger RNA markers for body fluid identification.Methods Mol. Biol. 2012; 830: 169-183
- Specific and sensitive mRNA biomarkers for the identification of skin in ‘touch DNA’ evidence.Forensic Sci. Int. Genet. 2012; 6: 548-558
- A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.Forensic Sci. Int. Genet. 2012; 6: 565-577
- Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis.Forensic Sci. Int. Genet. 2012; 6: 452-460
- mRNA profiling using a minimum of five mRNA markers per body fluid and a novel scoring method for body fluid identification.Int. J. Leg. Med. 2013; 127: 707-721
- Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification.PLoS One. 2014; 9: e100123
- Detection of epithelial cells in dried blood stains by reverse transcriptase-polymerase chain reaction.J. Forensic Sci. 1999; 44: 1232-1236
- Rapid and inexpensive body fluid identification by RNA profiling-based multiplex high resolution melt (HRM) analysis.F1000Research. 2013; 2 ([v1; ref status: indexed, http://f1000r.es/2hj]): 281https://doi.org/10.12688/f1000research.2–281.v1
- Multiplex mRNA profiling for the identification of body fluids.Forensic Sci. Int. 2005; 152: 1-12
- mRNA profiling for body fluid identification by multiplex quantitative RT-PCR.J. Forensic Sci. 2007; 52: 1252-1262
- Messenger RNA profiling: a novel method for body fluid identification by real-time PCR.Forensic Sci. Int. 2006; 157: 181-186
- Genome-wide mRNA profiling and multiplex quantitative RT-PCR for forensic body fluid identification.Forensic Sci. Int. Genet. 2013; 7: 143-150
- Identification of nasal blood by real-time RT-PCR.Leg. Med. 2012; 14: 201-204
- Evaluation of mRNA-based approach for identification of saliva and semen.Leg. Med. 2009; 11: 125-128
- mRNA-based skin identification for forensic applications.Int. J. Leg. Med. 2011; 125: 253-263
- MALDI-TOF mass spectrometrybased SNP genotyping.Pharmacogenomics. 2002; 3: 537-548
- Genotyping single-nucleotide polymorphisms by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry.J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2002; 782: 73-87
- Genotyping of dinucleotide tandem repeats by MALDI mass spectrometry of ribozyme-cleaved RNA transcripts.Nat. Biotechnol. 2001; 19: 877-880
- Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a revolution in clinical microbial identification.Clin. Microbiol. Infect. 2011; 16: 1614-1619
- Microbial fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) applications and challenges.Adv. Appl. Microbiol. 2010; 71: 149-184
- MALDI-TOF mass spectrometry for quantitative gene expression analysis of acid responses in Staphylococcus aureus.J. Microbiol. Methods. 2009; 78: 86-93
- Mass-spectrometry DNA sequencing.Mutat. Res. 2005; 573: 3-12
Sequenom, (http://www.sequenom.com/getdoc/197b98fa-93f7-40e8-9deb-a8dcfecf899e/iPLEX-brochure_web).
- Determination of an effective housekeeping gene for the quantification of mRNA for forensic applications.J. Forensic Sci. 2012; 57: 1051-1058
- Quantification on the lightcycler.in: Meuer S. Wittwer C. Nakagawara K. In Rapid Cycle Real-Time PCR, Methods and Applications. first ed. Verlag, Berlin2001: 21-34
- RNA/DNA co-analysis from human saliva and semen stains – results of a third collaborative EDNAP exercise.Forensic Sci. Int. Genet. 2013; 7: 230-239
- Protamine mRNA as molecular marker for spermatozoa in semen stains.Int. J. Leg. Med. 2003; 117: 175-179
- Erythroid-specific processing of human beta spectrin I pre-mRNA.Blood. 1994; 84: 1992-1999
- Selection of highly specific and sensitive mRNA biomarkers for the identification of blood.Forensic Sci. Int. Genet. 2011; 5: 449-458
- Housekeeping genes as internal standards in cancer research.Mol. Diagn. 2004; 8: 107-113
- Housekeeping gene sets facilitate the search for a suitable reference gene for relative quantification.Biochemica. 2002; 4: 9-11
- Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family.Mol. Biol. Evol. 1993; 10: 497-511
- Expression of protamine-1 and -2 mRNA during human spermiogenesis.Mol. Hum. Reprod. 2000; 6: 219-225
- iPLEX™ Assay: Increased Plexing Efficiency and Flexibility for MassARRAY® System Through Single Base Primer Extension with Mass-Modified Terminators. Sequenom® Application Note.2006 (Doc. No. 8876–006, R04 CO 060150 (http://agenabio.com/sites/default/files/iPLEX%20Application%20Note.pdf))
- MALDI-TOF mass spectrometry-based SNP genotyping.Methods Mol. Biol. 2003; 212: 241-262
- Sequenom® Application Note.2009 (Doc. No. 11555 R2.0 CO 090096 (http://agenabio.com/sites/default/files/iPLEX%20Gold%20Application%20Guide.pdf))
JSD Software, JMP software 10.0.1, Release 2, www.jmp.com.
- R: A Language and Environment for Statistical Computing.R Foundation for Statistical Computing, Vienna, Austria2009 (ISBN 3-900051-07-0)
- A novel method for real time quantitative RT-PCR.Genome Res. 1996; 6: 995-1001
- Quantification of mRNA using real-time RT-PCR.Nat. Protoc. 2006; 1: 1559-1582
- Mutation or loss of Wilms’ tumor gene 1 (WT1) are not major reasons for immune escape in patients with AML receiving WT1 peptide vaccination.J. Transl. Med. 2010; 8: 5
- Developmental validation of the quantifiler real-time PCR kits for the quantification of human nuclear DNA samples.J. Forensic Sci. 2005; 50: 809-825
- The real-time polymerase chain reaction.Mol. Aspects Med. 2006; 27: 95-125
- Comprehensive human adipose tissue mRNA and microRNA endogenous control selection for quantitative real-time-PCR normalization.Obesity (Silver Spring). 2011; 19: 888-892
- A time-course analysis of mRNA expression during injury healing in human dermal injuries.Int. J. Leg. Med. 2014; 128: 403-414
- Phosphoglycerate kinase 1 as a promoter of metastasis in colon cancer.Int. J. Oncol. 2013; 43: 586-590
- Phosphoglycerate kinase 1 a promoting enzyme for peritoneal dissemination in gastric cancer.Int. J. Cancer. 2010; 126: 1513-1520
- How specific are the vaginal secretion mRNA-markers HBD1 and MUC4?.Forensic Sci. Int.: Genet. Suppl. Ser. 2009; 2: 536-537
- mRNA profiling for the identification of blood – results of a collaborative EDNAP exercise.Forensic Sci. Int. Genet. 2011; 5: 21-26
- Illegitimate transcription: transcription of any gene in any cell type.Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2617-2621
- Expression of alpha-fetoprotein and prostate-specific antigen genes in several tissues and detection of mRNAs in normal circulating blood by reverse transcriptase-polymerase chain reaction.Jpn. J. Clin. Oncol. 1998; 28: 723-728
- Expression of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) in ileum and other extraprostatic tissues.Int. J. Cancer. 2005; 113: 290-297
- The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.Forensic Sci. Int. Genet. 2010; 4: 244-256
- Salivary transcriptome diagnostics for oral cancer detection.Clin. Cancer Res. 2004; 10: 8442-8450
- Characterization of RNA in saliva.Clin. Chem. 2006; 52: 988-994
- Quantitative real-time RT-PCR – a perspective.J. Mol. Endocrinol. 2005; 34: 597-601
- Implementation of RNA profiling in forensic casework.Forensic Sci. Int. Genet. 2013; 7: 159-166
- Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science.Int. J. Leg. Med. 2013; 127: 287-298
- Highly specific mRNA biomarkers for the identification of vaginal secretions in sexual assault investigations.Sci. Justice. 2013; 53: 14-22
Article info
Publication history
Published online: January 07, 2015
Accepted:
December 22,
2014
Received in revised form:
December 18,
2014
Received:
September 16,
2014
Identification
Copyright
Published by Elsevier Inc.
