Absolute and Relative Quantification of mRNA Expression (Transcript Analysis)

In this protocol, we describe a pipeline for transcript analysis in yeast via the quantification of mRNA expression levels. In the first section, we consider the well-established, proprietary Affymetrix GeneChip^� approach to generating transcriptomics data. In the next section, we concentrate on providing a detailed protocol for the validation of these data using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The protocol provides suggested examples of hardware, software, and consumables/reagents required to perform these experiments. There are of course many other options available using alternative approaches (or indeed suppliers), but this protocol is intended to provide an approach that is flexible, inexpensive, sensitive, and easy to use.