Quantitative real-time PCR for studies of microbial gene expression

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Modern day microbiology, like many life sciences, necessitates the application of molecular methods to address questions of gene diversity, abundance, and regulation. The polymerase chain reaction (PCR) is an everyday staple of the molecular microbiology tool chest as it allows for the amplification of individual gene targets for sequence analysis, cloning, nucleic acid hybridization, and many other applications. In the past few years, instrumentation has become readily available for real-time polymerase chain reaction, wherein one can measure the rate of single gene amplification during the PCR reaction in order to quantify the original number of a specific template molecule in a sample. Quantitative real-time PCR (qPCR) is rapidly becoming an indispensable technique and is even replacing such molecular biology classics as Northern blot hybridization for quantifying gene expression (once RNA molecules are converted into cDNA). My work involves comparing global gene expression (via cDNA array hybridization) in multiple species of ammonia-oxidizing bacteria under high and low oxygen tension and upon exposure to the nitrosating agents, nitrite and nitric oxide, to find genes involved in nitrosative stress response and nitrous oxide production. Once the genes are identified, their regulation and physiological function in wild-type and mutant backgrounds are carefully examined, and the same genes are used to examine levels of their expression in samples from natural ecosystems. All of these activities require qPCR to: 1) verify whole-genome expression data derived from microarray hybridization, 2) determine relative levels of gene expression in mutated versus wild-type bacteria, or bacteria exposed to different stimuli (e.g. nitrite, low oxygen, nitric oxide), and 3) determine the original amount of a gene template in a mixture of DNA or RNA extracted from environmental samples. qPCR is extremely powerful due to its low detection limit, the ability to amplify any part of a template, allowing one to query not just direct expression of a gene, but also the processing of RNA, and the ability to detect multiple templates in a single PCR reaction by using multiple fluorophores.