How do I choose the best internal control/reference/housekeeping gene for normalizing qPCR results?
IDT recommends that you test at least two, but preferably three, normalizing or housekeeping genes to ensure accurate internal controls. The best normalizing gene to use will depend on the species and conditions of the sample you will be testing. If you are unsure of the best normalizing gene to use, review the literature for the genes tested on samples with conditions similar to yours. When normalizing to a reference gene, it is very important that the reference gene is experimentally validated to ensure that it is an accurate measure against which to compare all other sample variations. A pilot study can be conducted to select the best set of reference genes out of a series of candidates. Analysis of reference gene stability can be performed with tools such as geNorm or qbase+ software (http://www.biogazelle.com/
). The reference genes should have stable mRNA expression and the amount of reference gene mRNA should be strongly correlated with the total amounts of mRNA in the samples. When using this method, it is critical that the reference genes used do not vary with experimental conditions. Normalized data is reported as a ratio of the mRNA concentration of the gene of interest to the mRNA concentration of the reference gene. This can be calculated by a comparative Cq
method or a standard curve method. For in depth discussion and recommendations on this topic, see the IDT webinar by Stephen Bustin & IDT, MIQE Guidelines: A Roadmap for Proper qPCR Experimental Design and Reporting