title: Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.
abstract: Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence.
authors.affiliation: National Center for Forensic Science, Orlando, FL 32816-2367, USA.
authors.forename: Erin K
authors.initials: EK
authors.lastname: Hanson
authors.affiliation: National Center for Forensic Science, Orlando, FL 32816-2367, USA ; Department of Chemistry, University of Central Florida, Orlando, FL 32816-2366, USA.
authors.forename: Jack
authors.initials: J
authors.lastname: Ballantyne
references.citation: Int J Legal Med. 2011 Mar;125(2):253-63
references.pmid: 21221983
references.citation: Invest Ophthalmol Vis Sci. 2007 Feb;48(2):486-90
references.pmid: 17251440
references.citation: Int J Legal Med. 2009 Jan;123(1):71-4
references.pmid: 18594850
references.citation: Hum Mutat. 2009 Jun;30(6):884-90
references.pmid: 19347964
references.citation: Forensic Sci Int Genet. 2011 Jan;5(1):21-6
references.pmid: 20457073
references.citation: Clin Chem. 2008 Oct;54(10):1648-56
references.pmid: 18676584
references.citation: Forensic Sci Rev. 2010 Jul;22(2):145-57
references.pmid: 26242593
references.citation: Int J Legal Med. 2013 Jul;127(4):707-21
references.pmid: 23254458
references.citation: Forensic Sci Int. 2006 Mar 10;157(2-3):181-6
references.pmid: 16289614
references.citation: Anal Biochem. 2009 Apr 15;387(2):303-14
references.pmid: 19454234
references.citation: Methods Mol Biol. 2012;830:169-83
references.pmid: 22139660
references.citation: Forensic Sci Int Genet. 2013 Jan;7(1):136-42
references.pmid: 22944353
references.citation: Forensic Sci Int. 2003 Aug 12;135(2):85-96
references.pmid: 12927408
references.citation: J Biomol Tech. 2009 Jul;20(3):160-4
references.pmid: 19568454
references.citation: Sci Justice. 2013 Mar;53(1):14-22
references.pmid: 23380057
references.citation: Int J Legal Med. 2003 Jun;117(3):175-9
references.pmid: 12799737
references.citation: Clin Chem. 2001;47(7):1183-9
references.pmid: 11427447
references.citation: Reproduction. 2002 May;123(5):621-31
references.pmid: 12006090
references.citation: Hum Mutat. 2009 Jun;30(6):860-6
references.pmid: 19418555
references.citation: Forensic Sci Int Genet. 2009 Mar;3(2):80-8
references.pmid: 19215876
references.citation: Forensic Sci Int. 2005 Aug 11;152(1):1-12
references.pmid: 15939171
references.citation: Electrophoresis. 2012 Jul;33(12):1736-45
references.pmid: 22740462
references.citation: J Forensic Sci. 2008 Mar;53(2):296-305
references.pmid: 18298493
references.citation: BMC Biotechnol. 2009 May 22;9:50
references.pmid: 19463169
references.citation: J Forensic Sci. 2012 Nov;57(6):1585-90
references.pmid: 22612601
references.citation: Forensic Sci Int Genet. 2012 Mar;6(2):274-6
references.pmid: 21612994
references.citation: Int J Legal Med. 2013 Jan;127(1):35-43
references.pmid: 22653424
references.citation: Clin Chem. 2004 Oct;50(10):1748-54
references.pmid: 15308590
references.citation: J Forensic Sci. 2011 Nov;56(6):1464-70
references.pmid: 21827476
references.citation: Hum Reprod. 1996 Oct;11 Suppl 2:124-33
references.pmid: 8982754
references.citation: Forensic Sci Int Genet. 2010 Jul;4(4):244-56
references.pmid: 20457026
references.citation: Forensic Sci Int Genet. 2012 Jan;6(1):70-80
references.pmid: 21459062
references.citation: Hum Mutat. 2009 Jun;30(6):891-8
references.pmid: 19370763
references.citation: Int J Legal Med. 2013 Mar;127(2):299-308
references.pmid: 22895803
references.citation: Cytogenet Genome Res. 2008;123(1-4):313-21
references.pmid: 19287170
references.citation: Biotechniques. 2009 Jan;46(1):58-60
references.pmid: 19301623
references.citation: Forensic Sci Int Genet. 2012 Sep;6(5):548-58
references.pmid: 22326111
references.citation: Int J Legal Med. 2012 Jan;126(1):55-62
references.pmid: 21626087
references.citation: Forensic Sci Int Genet. 2011 Nov;5(5):449-58
references.pmid: 20933484
references.citation: J Forensic Sci. 2007 Nov;52(6):1252-62
references.pmid: 17868268
references.citation: Forensic Sci Int Genet. 2013 Jan;7(1):116-23
references.pmid: 22909992
references.citation: Int J Legal Med. 2013 Mar;127(2):287-98
references.pmid: 22843116
references.citation: J Forensic Sci. 2002 Nov;47(6):1278-82
references.pmid: 12455649
references.citation: Int J Legal Med. 2014 Jan;128(1):33-41
references.pmid: 24052059
references.citation: Mol Cell Proteomics. 2012 Oct;11(10):1024-35
references.pmid: 22822186
references.citation: Int J Legal Med. 2010 May;124(3):217-26
references.pmid: 20145944
references.citation: Forensic Sci Int Genet. 2013 Feb;7(2):230-9
references.pmid: 23165093
references.citation: Int J Legal Med. 2008 Mar;122(2):135-42
references.pmid: 17579879
references.citation: Forensic Sci Int Genet. 2012 Jul;6(4):452-60
references.pmid: 22001154
references.citation: Forensic Sci Int Genet. 2012 May;6(3):419-23
references.pmid: 21903498
references.citation: Forensic Sci Int Genet. 2011 Nov;5(5):517-24
references.pmid: 21196138
references.citation: Am J Hum Genet. 2008 Mar;82(3):763-71
references.pmid: 18304490