I am going to study the expression of 8 target genes in respond to heat stress using qPCR. For example, comparing expression of gene 1 before and after treatment (not comparing gene 1 with gene 2). Do I need to obtain primers efficiency?
Thank you for your answers.
If you want a relative level of expression under the two conditions then you should be OK without doing an absolute calibration. This is under the assumption that although the absolute efficiency of your primers is unknonwn, that efficiency will be the same under the two conditions.
Yes. Primer efficiency of not only target but also normalization genes should be determined. If the efficiency of the primer pair is around 100%, then it means that amplification is doubled after every cycle. But efficiencies between 97-103 is acceptable, in my opinion.
Probably primer efficiencies are the same between your treated an non treaded samples.
Also consider potential batch effects if you run your samples accross multiple plates.
Thanks to all for the answers, I think it would not be wrong if I don't check the primer efficiency since I do not compare gene expressions with each other and I will run control and treatment on the same plate.
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