Housekeeping genes are genes needed for basic cell maintenance, such as metabolism, protein synthesis and cell structure, and they are usually expressed in most cells. In qPCR and RT-PCR they are used as reference genes (endogenous controls): the target gene's signal is normalized to the reference gene to correct for differences in RNA input, RNA quality and reverse-transcription efficiency between samples, for example with the ΔΔCt method.
No housekeeping gene is stable in every situation. Expression of common reference genes such as GAPDH and ACTB can change with tissue, cell type, developmental stage, disease or experimental treatment, and normalizing to an unstable reference gene can distort results. The MIQE guidelines therefore recommend validating reference genes for each experimental system and normalizing to more than one. Tools such as geNorm and NormFinder rank candidate genes by expression stability to help pick the best combination.
Our human, mouse and rat housekeeping gene primer sets let you test 8 to 10 commonly used reference genes from different functional pathways on your own samples, select the most stable ones and normalize to several housekeepers instead of one.
Which housekeeping genes are best for qPCR?
There is no universal best choice; it depends on your tissue and treatment. Test several candidates (our sets include ACTB, B2M, GAPDH, GUSB, HPRT1, PGK1, PPIA, RPL13A, TBP and TFRC) on your samples and choose the most stable with geNorm or NormFinder.
How many reference genes should I use?
The MIQE guidelines recommend normalizing to more than one validated reference gene rather than a single housekeeper.
Can these primers be used for RT-PCR?
Yes. They are real time PCR primer sets for measuring expression in cDNA made from RNA by reverse transcription (RT-qPCR).