For relative quantification of gene expression, it is important to choose a suitable gene to use as a reference (see table Housekeeping genes commonly used as endogenous references). The expression level of the reference gene must not vary under experimental conditions, or in different states of the same tissue or cell line (e.g., “disease” versus “normal” samples). The expression level of the reference RNA should also be approximately the same as the RNA under study. Reference RNA commonly used for relative quantification includes b-actin, b-2-microglobulin, peptidylprolyl isomerase A, and GAPDH mRNAs, and also 18S rRNA. b-actin mRNA is ubiquitously expressed and was one of the first RNAs to be used as a reference sequence. However, its transcription levels may vary and the presence of pseudogenes may mean that genomic DNA is detected during real-time PCR, leading to inaccuracies in quantification. GAPDH is a housekeeping gene commonly used as a reference for quantification of gene expression. GAPDH mRNA levels may vary between individuals, at different stages of the cell cycle, and following treatment with different drugs, making GAPDH unsuitable as a reference in some systems. As 18S rRNA is not an mRNA, its expression levels in the cell may not accurately reflect the cellular mRNA population. Therefore, a combination of genes may provide the most reliable reference for quantification studies.
Housekeeping genes commonly used as endogenous references