Chapter 9 – How Many Microorgansims Present? Quantitative Reverse Transcription PCR (QRT-PCR)

Abstract

Chapter 9 – How Many Microorgansims Present? Quantitative Reverse Transcription PCR (QRT-PCR)

Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells, and is produced by its corresponding gene to provide a ‘blueprint’ in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells, but also in inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oil field samples. When qRT-PCR is combined with primers and molecular probes for specific genes, the activity of microbial processes important in the oil field, such as Sulfate reduction, methanogenesis and nitrate reduction can be monitored.