i have used taqman Probes to quantify my gene of interest and house keeping gene. Can the relative quantification calulated by using delta delta ct?
Dear Shreyas,
Yes, you can! All you need is the CT value of your housekeeping gene(s) and gene(s) of interest(s) in both the control and the experimental samples.
The video, in attachement, gives you a nice overview of the process, calculations and assumptions.
Also, as with any qPCR experiements, I advocate to follow the MIQE guidelines to avoid common drawbacks and pitfalls of the qPCR method.
Best,
Nicolas
https://www.youtube.com/watch?v=tgp4bbnj-ng
http://www.rdml.org/clinchem.2008.112797v1.pdf
@ Franco
PCR efficiency is more important for delta delta CT (relative quantification) since both primer pairs (endogenous and target) need to be the ''same'' in order to compare them together.
However, when you use a standard curve method (absolute quantification), the efficiency will change the slope of the curve but not the direct comparison between two samples since it rely on one pair of primer running at the same efficiency.
Nicolas
thank you for your answers.
when performing such a qPCR, is it necessary to have both the gene of interest and house keeping gene on the same plate. What has to be done when your sample number is too high for gene of interest for one plate, can it be done on two plates and then compared?
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