Using endogenous control genes in real-time RT-PCR

When performing relative quantification of the expression of a target gene, it is important to choose a suitable gene for use as a reference or endogenous control. An endogenous control gene is a gene whose expression level should not differ between samples, such as a housekeeping or maintenance gene. Comparison of the C_T value of a target gene with that of the endogenous control gene allows the gene expression level of the target gene to be normalized to the amount of input RNA or cDNA. This is done without determining the exact amount of template used in the reaction. The use of an endogenous control gene corrects for variation in RNA content, variation in reverse-transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA sample, variation in nucleic acid recovery, and differences in sample handling. Genes commonly used as references are shown in the tables below.