Measurements of mRNA expression levels—whether by Northern analysis, ribonuclease protection, or real-time quantitative PCR—are usually standardized by comparing the data to that obtained for an internal or endogenous reference gene. Housekeeping genes such as beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are most often used because their expression levels are expected to remain constant under different treatment conditions. Unfortunately, this assumption is not always valid, and results based on housekeeping genes alone can be biased (1). A better method is to normalize your data using our qPCR Human Reference cDNA, the only total cDNA control derived entirely from human tissues (2).
qPCR Human Reference cDNA is the ideal control for comparing data from different quantitative PCR (qPCR) experiments. Because it is prepared from a total RNA pool collected from several different tissues, our qPCR Human Reference cDNA provides broad gene coverage, as shown by microarray analysis of the RNA starting material. RNA, and therefore cDNA, prepared from whole tissues provides better gene representation with less variation than RNA made from cell lines (data not shown). Moreover, PCR analysis shows that our Total RNA is virtually free of genomic DNA (3). This allows for a more accurate measurement of transcript copy number. Both high and low abundance genes are well represented allowing preparation of a wide range of serially diluted standards for each qPCR assay. Lot-to-lot variation of Reference cDNA is minimal because the RNA source is prepared on an industrial scale.