I would like to determine several gene expressions for 4 different samples using qPCR with SYBR Green. Is it possible to run all of them in one plate regard to primers Tm differences? Would run gradient reaction helps to overcome Tm issue?Any help would be appreciated. Thanks.
Hi
Yes, as long as you don't have drastically different Tms or product sizes you can run them all together. This is a totally commonplace lab practice. You are comparing the same gene/primers between samples so the PCR efficiency is the same across the samples.
Hi Ahmed,
I agree with Kenneth that it would be acceptable to check several gene expressions on your samples. I seldom check one gene, thought I usually have more samples than I can run on one plate for one gene.
Might I suggest that you use two (or more) internal references when checking more than one gene. Certain tissues from the same source that I have checked give different responses to an internal reference. Consequently, I might use 18S along with GAPDH in certain normal-abnormal/tumor tissues or tissues from different organs from the same source.
If your product sizes are very similar (should be easily achieved using Primer3 Plus parameters or alternate choices for primers), you should be able to account for any Tm differences. I suspect that you would not find the Tm a problem. I highly recommend having a control sample so that the software in your real time apparatus can assist you with the analysis.
Best of luck,
Dan
Thank you very much for everyone. Your comments and suggestions are really helpful.
Thanks again
Ahmed,
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