Reference genes

Also called housekeeping genes, they are usually genes essential for the cell because they are needed for basic cellular functions. Reference genes are expected to be expressed at relatively constant levels in most of the cells of an organism under normal and diseased conditions.

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

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 (Glyceraldehyde 3-phosphate dehydrogenase), and ACTB (the gene that codifies beta-actin). 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.