I have two types of template (RNA) derived from two different types of tissue (normal and tumor). Which one I should add to well with endogenous control primers/probe (GAPDH) at qPCR plate? I know that there is no difference because it is a control gene (it is suggested that its expression will be equal in both types of tissue, but, nevertheless, I would like to know which source of template is usually used in this case (template from normal or tumor sample)? And, additionally, should I make all templates' concentrations equally (in every well at plate) before start of qPCR (TaqMan Gene Expression Assay)?
Please read about qPCR experimental design before you start pipetting.
You have to use the endogenous control for ALL of your samples. You cannot assume that the expression is "the same", the entire purpose of that control is to be able to measure the expression & use that as a reference to compare expression between samples & treatments.
No, you do not need to make the template the same concentration. That's the reason you use the controls.
Good luck!
The "endogenous control" is used to calibrate against RNA isolation efficiency and sample loading differences between your samples, so you need it in each of your samples. It's not to *show* that the expression of that gene is equal in all samples - the assumption of equal expression is *used* to allow that calibration.
Also note that (1) using a single control is not state of the art and prone to errors and bias, and that (2) GAPDH is known to be regulated under oxidative stress (hypoxia) and that hypoxia is a often a key feature in the development of tumors (hence GAPDH might *not* be a valid control).
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