We have developed a two-step, real-time, quantitative assay, using the polymerase chain reaction with reverse transcription (RT-PCR) and based on SYBR Green I monitoring of PCR product accumulation, for quantification and normalization of gene expression levels. Because housekeeping gene expression can vary considerably among cell types or experimental conditions, our procedure uses multiple internal control genes for more accurate normalization of expression data, compared with traditional use of only one housekeeping gene. Owing to extensive accumulation of primer-dimers when no template control is used during one-step RT-PCR, we introduced a two-step protocol to eliminate this problem. This study further illustrates the prerequisite of DNase treatment of RNA samples before complementary DNA synthesis. The treatment also results in a significantly facilitated primer design for RT-PCR, as the positions of the primers are no longer important to control for genomic contamination. Real-time quantitative RT-PCR of DNase-treated samples and normalization using multiple internal controls is the method of choice for sensitive, accurate and large-scale measurements of gene expression levels.