Molecular Biomarkers: The Development of MRNA Multiplex RT-PCR assay for the Definitive Identification of Semen

Abstract

Background: Semen is one of the most common body fluids found at the crime scene. Protamine-1 (PRM1), protamine-2 (PRM2) and semenogelin-1 (SEMG1) are specific mRNA markers found only in semen so if it is possible to detect these markers in a forensic specimen by RT-PCR, this could be helpful to verify the presence of semen. Aim of the work: Identification of semen in forensic samples by molecular assessment of specific RNA biomarkers. Subjects and methods: Semen samples were obtained from 50 inpatients and outpatients of the andrology department that were divided into 3 groups including: normozoospermic (21 specimen), oligozoospermic (11 specimen) and azoospermic (18 specimen) groups and 10 blood samples were taken from the normozoospermic patients to assess the presence of the markers in blood. The next steps of work were RNA extraction from semen and blood, PCR amplification of genes, detection of the amplified gene using agarose gel electrophoresis and performing gene expression analysis of the 3 genes using quantitative RT-PCR. Results: PRM1, PRM2 and SEMG1 were only detected in semen samples and were totally absent in blood samples. PRM1 was the most specific and reliable marker for semen identification followed by PRM2 and SEMG1 which was the least specific. These markers are not only used for identification but also for detection of the semen profile of the suspect. There was a direct positive correlation between sperm count and RNA expression. Conclusion: The analysis of the RNA profile in a sample can uniquely identify the fluid or tissue of origin. RT- PCR is valuable in semen identification by gene expression analysis of PRM1, PRM2 and SEMG1. PRM1 was the most specific and reliable marker used for semen identification.