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Endogenous Controls in qPCR - Ask TaqMan #24
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Endogenous Controls in qPCR - Ask TaqMan #24
Submit your question and we might make a video out of it: http://bit.ly/1cgFftk If you are studying gene expression by real-time PCR, then you are likely using relative quantitation or comparative Ct method. This method of analysis requires the use of an endogenous control, that is, a gene which does not change expression across different samples, treatments, or time points. This stable expression is required in order to provide a reliable basis for gene comparison. So, you may have asked yourself a question like the one we received from Wade Powell from Kenyon College, "What's the best way to select a valid endogenous control for relative qPCR?" 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. Since there is no way to anticipate how a particular treatment or diseased state will affect gene expression, the only way to definitively identify the right control is to simply perform a qPCR run with your samples. You will need to choose some candidate genes and check their expression.
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