Quantification of RNA degradation by semi-quantitative duplex and competitive RT-PCR: a possible indicator of the age of bloodstains?

Abstract

In vitro RNA degradation is a complex and non-linear process which can serve as indicator for the quality and age of stains. We have developed a semi-quantitative duplex reverse transcription-polymerase chain reaction (RT-PCR) assay which, in combination with competitive RT-PCR using an external standard, allows quantification of RNA degradation levels. Using this method, we have investigated 106 bloodstains stored up to 15 years. The distribution of the peak area quotients of standard and target messenger-RNA (mRNA) as measured by laser-induced fluorescence capillary electrophoresis was closely correlated with the age of the samples. Further statistical analysis showed that bloodstains with age differences of 5 years and more exhibit statistical significant variances in peak area quotients of both housekeeping genes included in this study, β-actin and cyclophilin. This can be of value when evidence from old cases is re-investigated. Our data show, that, although RNA continues to be degraded in dried bloodstains, mRNA suitable for RT-PCR can be isolated from samples stored for at least 15 years.