Development of Highly Sensitive and Specific mRNA Multiplex System (XCYR1) for Forensic Human Body Fluids and Tissues Identification

Abstract

The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.

Differential genes expression biomarkers for menstrual and peripheral blood stains analysis

“…This is consistent with other studies where MMP7 and MMP11 appeared equally useful in the determination of menstrual secretion although MMP7 seems to be more sensitive; as higher signals were obtained when compared to MMP11 throughout menstrual days (Hass et al, 2009;Song et al, 2015;Lindenbergh et al, 2012;Xu et al, 2014).…”

Forensic Genetics and Genotyping

“…In 2003, using standard RT-PCR and gel electrophoresis, Juusola and Ballantyne reported that mRNAs specific for saliva and semen can be detected in stains as old as 10 weeks (15,57). The mRNAs have been used for identification of human body fluids or tissues, which is very useful for forensic investigations to identify the origin of biological samples (3,58). In 2005, Juusola and Ballantyne demonstrated the distinction of stains originating from different body fluids such as blood, semen, saliva and vaginal secretions (59).…”

Tissue Typing by Means of PCR and Capillary Electrophoresis: New Aspects, Reality and Issues

“…В целом, как следует из разных публикаций по ра-боте с серийными разведениями, мультиплексный ПЦР на основе мРНК обладает высокой чувствительностью и может улавливать до 5-8 пг РНК, что соответствует количеству РНК в одной клетке [11,12].…”

“…В последующие годы за счет открытия и внедре-ния новых маркеров появились новые версии панели «CellTyper», равно как и её аналоги [11], и настоящая, третья версия (CellTyper -v.3) включает в себя 19 мРНК-маркеров (см. табл.…”

“…С другой стороны, это открывает возможность отечественным исследователям разрабатывать собственные панели и наборы. Примером такой изменчивости может слу-жить разновидность панели «CellTyper» для азиатской популяции -«XCYR1» [11].…”