Evaluation of the inclusion of circular RNAs in mRNA profiling in forensic body fluid identification

The use of messenger RNA (mRNA) profiling is considered a promising method in the identification of forensically relevant body fluids which can provide crucial information for reconstructing a potential crime. However, casework samples are usually of limited quantity or have been subjected to degradation, which requires improvement of body fluid identification. Circular RNAs (circRNAs), a class of products from the backsplicing of pre-mRNAs, are shown to have high abundance, remarkable stability, and cell type-specific expression in human cells. In this study, we investigated whether the inclusion of circRNAs in mRNA profiling improve the detection of biomarkers including δ-aminolevulinate synthase 2 (ALAS2) and matrix metallopeptidase 7 (MMP7) in body fluid identification. The major circRNAs of ALAS2 and MMP7 were first identified and primer sets for the simultaneous detection of linear and circular transcripts were developed. The inclusion of circRNAs in mRNA profiling showed improved detection sensitivity and stability of biomarkers revealed by using serial dilutions, mixed samples, and menstrual bloodstains as well as degraded and aged samples. Therefore, the inclusion of circRNAs in mRNA profiling should facilitate the detection of mRNA markers in forensic body fluid identification.

[1]  Woo Ick Yang,et al.  Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA , 2013, International Journal of Legal Medicine.

[2]  W Parson,et al.  RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise. , 2012, Forensic science international. Genetics.

[3]  W Bär,et al.  mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. , 2009, Forensic science international. Genetics.

[4]  Sebastian D. Mackowiak,et al.  Circular RNAs are a large class of animal RNAs with regulatory potency , 2013, Nature.

[5]  Julia Salzman,et al.  Cell-Type Specific Features of Circular RNA Expression , 2013, PLoS genetics.

[6]  Cordula Haas,et al.  mRNA profiling using a minimum of five mRNA markers per body fluid and a novel scoring method for body fluid identification , 2013, International Journal of Legal Medicine.

[7]  W. Yang,et al.  DNA methylation-specific multiplex assays for body fluid identification , 2012, International Journal of Legal Medicine.

[8]  Titia Sijen,et al.  A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces. , 2012, Forensic science international. Genetics.

[9]  Li Yang,et al.  Regulation of circRNA biogenesis , 2015, RNA biology.

[10]  Sven Diederichs,et al.  Argonaute proteins regulate microRNA stability: Increased microRNA abundance by Argonaute proteins is due to microRNA stabilization , 2011, RNA biology.

[11]  Titia Sijen,et al.  Development of a mRNA profiling multiplex for the inference of organ tissues , 2013, International Journal of Legal Medicine.

[12]  Oh-Hyung Kwon,et al.  Microarray screening and qRT‐PCR evaluation of microRNA markers for forensic body fluid identification , 2014, Electrophoresis.

[13]  Charles Gawad,et al.  Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types , 2012, PloS one.

[14]  Á. Carracedo,et al.  RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise. , 2013, Forensic science international. Genetics.

[15]  C. Courts,et al.  Identification of gunshots to the head by detection of RNA in backspatter primarily expressed in brain tissue. , 2014, Forensic science international.

[16]  Oh-Hyung Kwon,et al.  Identification of body fluid-specific DNA methylation markers for use in forensic science. , 2014, Forensic science international. Genetics.

[17]  Jack Ballantyne,et al.  Multiplex mRNA profiling for the identification of body fluids. , 2005, Forensic science international.

[18]  N. Sharpless,et al.  Detecting and characterizing circular RNAs , 2014, Nature Biotechnology.

[19]  Meng-Han Lin,et al.  Degraded RNA transcript stable regions (StaRs) as targets for enhanced forensic RNA body fluid identification. , 2016, Forensic science international. Genetics.

[20]  W Parson,et al.  mRNA profiling for the identification of blood--results of a collaborative EDNAP exercise. , 2011, Forensic science international. Genetics.

[21]  L. Ripani,et al.  Molecular identification of vaginal fluid by microbial signature. , 2012, Forensic science international. Genetics.

[22]  Cornelius Courts,et al.  Differentiation of five body fluids from forensic samples by expression analysis of four microRNAs using quantitative PCR. , 2016, Forensic science international. Genetics.

[23]  Iva Gomes,et al.  A 17-month time course study of human RNA and DNA degradation in body fluids under dry and humid environmental conditions , 2016, International Journal of Legal Medicine.

[24]  Titia Sijen,et al.  Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling. , 2016, Forensic science international. Genetics.

[25]  Michael K. Slevin,et al.  Circular RNAs are abundant, conserved, and associated with ALU repeats. , 2013, RNA.

[26]  Ling-Ling Chen,et al.  Complementary Sequence-Mediated Exon Circularization , 2014, Cell.

[27]  SallyAnn Harbison,et al.  The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids. , 2010, Forensic science international. Genetics.

[28]  J. Ballantyne,et al.  Recovery and Stability of RNA in Vaginal Swabs and Blood, Semen, and Saliva Stains , 2008, Journal of forensic sciences.

[29]  D. Tollervey,et al.  The Many Pathways of RNA Degradation , 2009, Cell.

[30]  N. Rajewsky,et al.  circRNA biogenesis competes with pre-mRNA splicing. , 2014, Molecular cell.

[31]  D. Bartel,et al.  Expanded identification and characterization of mammalian circular RNAs , 2014, Genome Biology.

[32]  W Bär,et al.  Selection of highly specific and sensitive mRNA biomarkers for the identification of blood. , 2011, Forensic science international. Genetics.

[33]  Kelly Virkler,et al.  Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. , 2009, Forensic science international.

[34]  Developed and evaluated a multiplex mRNA profiling system for body fluid identification in Chinese Han population. , 2015, Journal of forensic and legal medicine.

[35]  Tim Schneider,et al.  Exon circularization requires canonical splice signals. , 2015, Cell reports.

[36]  Jack Ballantyne,et al.  mRNA Profiling for Body Fluid Identification by Multiplex Quantitative RT‐PCR * , 2007, Journal of forensic sciences.

[37]  James M Robertson,et al.  Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis. , 2012, Forensic science international. Genetics.