Analysis of mRNA from human heart tissue and putative applications in forensic molecular pathology

Abstract

The usefulness of post-mortem mRNA analysis and its potential applications in forensic casework is currently of interest, especially because of several factors affecting the quality of RNA samples that are not practically predictable. In fact, post-mortem RNA degradation is a complex process that has not been studied systematically. The purpose of this work is to establish whether RNA analysis from post-mortem heart tissue could be used as a forensic tool to investigate the cause of death, with special regard to those cases where a cardiac disease is suspected as the manner of death. We analysed heart tissue from 16 individuals with normal cardiac function, 9 with long post-mortem intervals (L-PMI) and 7 from organ donors with very short PMIs (S-PMIs). Right ventricle tissue was homogenised, and the RNA was isolated and reverse transcribed. The resulting cDNA was used in real-time PCR reactions to quantify the gene expression of beta-glucuronidase (GUSB), Nitric Oxide Synthase 3 (NOS3), Collagen 1 (COL1A1) and Collagen 3 (COL3A1). The percentage of samples with high-quality RNA was higher in samples with S-PMI (7 out of 7) than in samples with L-PMI (4 out of 9, p<0.05). No differences in PMI time or cause of exitus were found between samples with degraded or non-degraded RNA in the L-PMI group. When comparing mRNA levels in samples with non-degraded RNA, we found similar values between the L-PMI and S-PMI groups for GUSB, COL1A1 and COL3A1. The NOS3 gene expression in the L-PMI subgroup was less than half that in the S-PMI. These results suggest that high-quality mRNA can be extracted from post-mortem human hearts only in some cases. Moreover, our data show that mRNA levels are independent from the PMI, even though there are mRNAs in which the expression levels are very susceptible to ischemia times. Clear knowledge about the relationship between mRNA integrity and expression and PMI could allow the use of several mRNAs as forensic tools to contribute to the determination of the cause of death with special regard to cardiovascular diseases.