i am looking for quantifying the transcript level of certain gene in the presence and absence of the drug pressure. The Ct value of my target gene is comparatively higher in the presence of the drug. However this increased Ct value is less than the Ct value of the reference gene.
Is this perfect? Can I proceed for delta delta Ct value calculation?
Yes I think it is fine. treatment of your drug increased the expression level of your target gene.it is not necessary that it should cross the expression level of housekeeping gene to considered as induced.Housekeeping gene is used for data normalization.
Hello Jochen
Thanks for for your answer. Actually I was confused regarding the delta delta Ct value calculation.
I have made proper analysis for the selection of reference gene. It was based on previous study.
I agree with Jochen. Regarding the delta delta ct calculation, the first delta ct is between your house-keeping gene and other genes in your list under different conditions. The house-keeping gene should not change under different treatment conditions and its expression is used to check your technical variation. The second delta ct is between your treatment and control conditions.
Best practices for RT-qPCR dictates that you need multiple reference genes to ensure that they are stable. Multiple reference genes can be evaluated using several algorithms but I really like geNorm. Check out the helpful MIQE guidelines for proper use of RT-qPCR. doi: 10.1373/clinchem.2008.112797
Dear Fraser Clark thanks for your suggestions. I will definitely go through MIQE guidelines.
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