Also called housekeeping genes, they are usually genes essential for the cell because they are needed in the sustenance of basic cellular function. Reference genes are expected to be expressed at relatively constant levels in most of the cells of an organism under normal and diseased conditions. Reference genes may vary depending on experimental conditions, therefore you always need to test several and assess which are the most stable ones in your settings. Typical reference genes are GAPDH and ACTB.

Differences in RNA expression level between two samples can be the result of both true biological as well as technical variation. It is very important to minimize the technical variation by using proper normalization to stably proved reference genes which will allow a better appreciation of the true biological variation. The reference genes are used for normalization of theqPCRdata. In fact many factors can contribute to variability in the sample analysis. This variability is often related to the reverse transcription efficiency for each sample. More contaminants remain in your RNA sample less efficient is the reverse transcription reaction and this effect varies between your samples.

There are several programs that can test the stability of potential reference genes (e.g. GeNorm, NormFinder) . These programs can be used to choose the best 2-3 reference genes for your project.