Real-time PCR detection of five different “endogenous control gene” transcripts in forensic autopsy material

Abstract 

Relative quantification of mRNA using quantitative real-time reverse transcription (RT)-PCR is a commonly used method for analysis and comparison of gene expression levels. This method requires a normalisation of data against expression levels of a control gene. In the past, several ubiquitously expressed genes were used as such endogenous controls. When working with human tissue samples obtained during autopsy one has to deal with postmortem intervals of usually more than 10h. The aim of this study was to investigate whether commonly used endogenous control genes show stability over various postmortem intervals. For this purpose, RNA was extracted from three different human tissues of five postmortem intervals ranging from 15 to 118h. The C_t values from five commonly used endogenous control genes – β-actin, B2M, CyPA, TBP, and UBC – were obtained by real-time RT-PCR.

Results revealed a relatively high stability of C_t values in skeletal muscle tissue regarding different postmortem intervals. In heart and brain tissues, all endogenous controls were found to be highly variable. B2M appeared to be the least unstable control in this set. Nevertheless, all endogenous controls showed variability in their expression levels regarding both the stability among different tissues and different postmortem intervals.

Data obtained in the present study show that postmortem mRNA degradation is a complex process, and that the use of one single endogenous control in gene expression studies of postmortem tissue would lead to erroneous data interpretation. Further studies on this topic should be performed in the future including an increased number of well documented samples.

Keywords: Quantitative real-time RT-PCR, Forensic autopsy material, Data normalisation, Endogenous control gene