I have three target genes (hepcidin, MHC and IL), all of them are expressed from the same tissue (kidney). I will quantify their relative gene expression (qPCR) using beta actin as my internal control. Given that all these four genes (target genes plus internal control) have the same thermal profiles, can I run my internal control once and use its cp value to compute the relative expression of my target genes? I am thinking to do this in order to save reagents since they were all came from the same tissue and same thermal profile.
Maybe I did not get 100% what you really want - but when you want to go for relative quantification of your genes, you have to run your samples with your reference gene as well as target gene(s) in the same PCR. So, e.g. in a 96 well plate maybe the first 4 or 6 wells should contain your beta-acin, the next then hepdicidin, the next ones then MHC and than IL. When designing your primers for your PCR you should ensure that all primers work with the same annealing temperature so that you can run them all in one PCR.
But in each an every PCR plate you run you have to put also your reference gene as the efficiency of PCR might change a bit from plate to plate and the reference gene is also some control for this.
Hi Yasser, what is your technical approach? Are you analyzing by the comparative Ct-method? Do you use TaqMan probes or SYBR green? However, if you use probes and run your control as well as your target in one well, you should not use the Ct of one reaction for calculations of another target, since the two reactions occuring in one well could specifically interfere.
If you use SYBR green (control and target in different wells), you can of course run your control once and compare it to all your targets. However, if you apply the second approach, you must be very accurate at pipetting (amounts of RNA/cDNA must be quite the same in each of the compared wells, since you do not only compare results measured within one well). Moreover, if you apply delta delta Ct without using pre-designed probes, you have to check, whether the efficiency of your target and your control PCR is approx. the same (by comparing your delta Ct at different dilutions of a sample).
Foy SYBR green PCR once quantifying the control is the logical way and doing it several times doesnt make much sense. The taqman assays give u additional opportunity to multiplex and make sure that u normalize also ur pipetting errors everytime u quantify target genes. I dont find it anyway wrong to use taqman values of control genes to normalize those for SYBR quantified targets (if ur idea is only relative quantification)
Hi to all, I would like to thank you for answering my query. I will consider all your points before I start my experiment.
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