Studies on RNA integrity and gene expression in human myocardial tissue, pericardial fluid and blood, and its postmortem stability.

Abstract

Analyses of gene expression of ischemic myocardial injury and repair related proteins has been carried out for the first time in samples from five specific sites of the myocardium, pericardial fluid and blood from thirty cadavers in relation to post-mortem interval (PMI). RNA integrity was evaluated by RNA integrity number (RIN), with values ranging from 6.57 to 8.11; sufficiently high levels of integrity to permit further gene amplification. No significant correlations between RIN and PMI in any samples were detected. Prior to target gene expression analysis, a normalization strategy was carried out to assess candidate reference gene stability, involving the analysis and comparison of four common housekeeping genes (Glyceraldehide-3-phosphate dehydrogenase, beta-actin, TATA box binding protein and Cyclophilin A). Gene expression of cardiac troponin I (TNNI3), myosin light chain 3 (MYL3), matrix metalloprotease 9 (MMP9), transforming growth factor beta 1 (TGFB1), and vascular endothelial growth factor A (VEGFA) in myocardial zones and body fluids were subsequently studied by real-time quantitative PCR. Expression levels of all the proteins studied in cardiac zone samples were similar. No statistical differences for expression were detected among proteins taken from any myocardial area. No significant differences were detected for TNNI3 and TGFB1 gene expressions when compared with samples at or under 12h-PMI or over 12h-PMI. However, differences in MYL3, MMP9, and VEGFA gene expression in body fluids were found at PMI periods of over 12h. These interesting results may contribute to the refinement of current knowledge regarding cardiac metabolism and improve understanding of the underlying mechanisms involved in myocardium ischemia and its repair.