Quantitative gene expression analysis is fundamental to many experimental protocols and hypothesis testing in scientific research. The most popular currently used method to measure the expression level of specific genes in biological samples is real-time quantitative polymerase chain reaction (PCR). The method itself has become routine in many laboratories however stringent protocols and careful planning are required to for the generation of meaningful data. Many variations to these protocols are described in the literature. We describe here the methods used in our laboratory that have been compiled following many hours of troubleshooting and in our view they are robust protocols, providing solid data. The protocols are applicable to tissue culture cells where acute changes in gene expression are routinely observed following exposure to chemical or environmental stimuli, as well as tissue samples where gene expression is altered as a result of disease processes and interventions.