Effects of PCR inhibitors on mRNA expression for human blood identification

Detection of body fluid-specific mRNAs, such as those specific for blood, using real-time polymerase chain reaction (PCR) has become a useful tool in forensic science. Blood stains often contain PCR inhibitors that may be co-extracted with RNA and adversely affect PCR. The effects of inhibitors on the detection of mRNA markers for blood identification, namely, hemoglobin beta (HBB) and actin beta, were examined herein. Inhibitors were added to a real-time PCR mix, reverse transcription mix, and blood samples before RNA extraction, and the following parameters: Ct, delta Ct (dCt), and melting temperature (Tm) values, were monitored. Hematin, humic acid, indigo carmine, and tannic acid were used as PCR inhibitors. The results showed that Ct values for HBB in samples containing inhibitors in their real-time PCR mix increased in a concentration-dependent manner, and were undetectable at higher concentrations. Moreover, Ct values for HBB in tannic acid-spiked samples reached a maximum once, and inhibition decreased at higher concentrations. dCt values increased in hematin-spiked samples, but decreased in tannic acid-spiked samples. Tm values decreased following the addition of each inhibitor. The reverse transcription reaction was scarcely inhibited at concentrations that markedly affected realtime PCR. The complete removal of inhibitors added to blood was difficult. However, the observed inhibitory effects were weaker than those when inhibitors were added to the PCR cocktail. PCR inhibition was effectively reduced by repurification of complimentary DNA with DNA extraction kits. These results will assist examiners in deducing contaminating inhibitors and selecting an appropriate method to remove them.