An Internal Reference Technique for Accurately Quantifying Specific mRNAs by Real-Time PCR with Application to the tceA Reductive Dehalogenase Gene

Abstract

The accuracy of mRNA quantification by reverse transcription (RT) in conjunction with real-time PCR (qPCR) is limited by mRNA losses during sample preparation (cell lysis, RNA isolation, and DNA removal) and by inefficiencies in reverse transcription. To control for these losses and inefficiencies, a technique was developed that utilizes an exogenous internal reference mRNA (ref mRNA) along with mRNA absolute standard curves. The technique was applied to quantify mRNA of the trichloroethene (TCE) reductive dehalogenase-encoding tceA gene in an anaerobic TCE-to-ethene dechlorinating microbial enrichment. Compared to RT-qPCR protocols that utilize DNA absolute standard curves, application of the new technique increased measured quantities of tceA mRNA by threefold, demonstrating a substantial improvement in quantification. The technique was also effective for quantifying the loss of mRNA during specific steps of the sample processing protocol. Analysis revealed that the efficiency of the RNA isolation (56%) step was significantly less than that of the cell lysis (84%), DNA removal (93%), and RT (88%) steps. The technique was applied to compare the effects of cellular exposure to different chlorinated ethenes on tceA expression. Results show that exposure to TCE or cis-1,2-dichloroethene resulted in 25-fold-higher quantities of tceA mRNA than exposure to vinyl chloride or chlorinated ethene starvation.