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 substantia

SUBMITTER: Johnson DR 

PROVIDER: S-EPMC1169012 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature