Endogenous Controls in qPCR

Choosing an Endogenous Control for Gene Expression Analysis Using Real-Time PCR

Relative gene expression is one of the most common applications that researchers perform on their real-time instruments. Whenever we do a gene expression experiment, we need at least two gene-specific assays. One for our gene of interest, and one for an internal control gene, also sometimes referred to as a “normalizer,” “housekeeping gene,” or “endogenous control.”
An endogenous control gene shows expression levels that are relatively constant and moderately abundant across tissues, cell types, and treatment protocols. Normalization to endogenous control genes is currently the most accurate method to correct for potential biases that are caused by:

  • Sample collection
  • Variation in the amount of starting material
  • Reverse transcription (RT) efficiency
  • Nucleic acid (RNA/DNA) preparation and quality

To identify the best endogenous control, we need to find a gene that does not change expression across our different samples. Stable expression is defined as small variations, between 0-0.5 Cts, among the samples for the endogenous control gene. Keep in mind that a difference of one cycle equates to a twofold difference in initial template. Furthermore, a control with ∆CT values that vary over a two-cycle range would have nearly a fourfold difference in expression levels. If we were to select a normalizer gene whose expression varied by two or four-fold between samples, then the final fold calculations would be in error by this same factor.

Here are some easy steps to follow to find candidate genes and check their expression:

  1. Identity your candidate endogenous control genes. If you are using human or mouse samples, you can use our application note. Also, you can check in the literature or simply test some common controls. You can also try a larger test, such as one of the endogenous control panel.
  2. Purify the RNA using the same method across all your samples.
  3. Quantify and use the same amount of RNA from each sample of your RT reaction.
  4. Test your candidate endogenous control genes in your qPCR reaction using the same volume of cDNA in each reaction.
  5. Check the ∆CT between samples for each candidate endogenous control gene. The best control would have dCT as close to zero as possible. Our relative quantification software will score the candidate control. The lowest score indicating the most appropriate control.