Quantification of Host and Phage mRNA Expression During Infection Using Real-Time PCR

Real-time, or quantitative PCR, is a valuable technique useful in bacteriophage research to quantify the abundance of phage or host gene transcripts. It can be used during the infection cycle both to monitor the expression of individual viral transcripts and to compare relative gene expression levels throughout the infection cycle. It is fairly economical to conduct and is useful in bacteria–phage systems where obtaining high yields of RNA is problematic. To perform real-time PCR, it is simply necessary to know the DNA sequence of the genes to be monitored, to have accurately quantified mRNA good quality cDNA, and access to a light-cycler. Although this chapter briefly reviews the basic principles of real-time PCR, the emphasis is on aspects of technique that are specific to the study of bacteriophage transcriptomics. These include (1) the selection of the target gene, (2) the choice of calibrator and reference genes, (3) RNA isolation for cDNA synthesis and (4) subsequent analysis of samples. This chapter should also be useful to those wishing to amplify genes from other types of templates such as metagenomic DNA or RNA extracted either from filtered samples or from agarose gels.