Evaluating Gene Expression in Ustilago maydis Using RT-qPCR

Gene expression analysis detects changes in the transcript level of a gene in response to a specific condition. Analysis of these changes can reveal links to changes in the growth, development or physiology of an organism. In Ustilago maydis, reverse transcriptase quantitative PCR (RT-qPCR) has primarily been used to examine the changes in messenger RNA (mRNA) levels. This chapter outlines a method for assessing gene transcript level changes using RT-qPCR and the comparative C_T (2^-ΔΔCT) method. Successful RT-qPCR experiments rely on several factors including primer design and primer efficiency. In addition, the comparative C_T method reports relative gene expression levels, which relies on the selection of a reference gene with stable mRNA levels in the cell types being examined. The presented protocol includes considerations for primer design, validation of reference genes, and assessing primer efficiency. Guidance is provided on synthesizing cDNA, setting up RT-qPCR reactions, and analyzing data using the comparative C_T method.