Hello, I am doing qPCR experiments on RNA extracted from mouse spleens and ganglia. At first I found that beta actin is expressed from 28th cycle. I tried to lower the hybridization temperature from 65C to 62C and beta actin is expressed from the 22nd cycle. when I lowered the temperature to 60 the number of cycles for beta actin increased again to 26 cycles. I ask can beta actin not be expressed until the 22nd cycle? and thank you very much
The Ct value depends on the efficiency of the reaction and the amount of starting template.
Assuming you used the exact same amount of the exact same tube of cDNA & other reagents, then you are seeing effects of how changing the annealing temp changes the efficiency of the reaction.
What is the calculated efficiency from your standard curve?
Hello.
Please, check the PCR efficiency at all three temperatures mentioned:
Use serial dilutions of your cDNA and run these wells as "Standard" entering dilution rate in your plate data.
Hope this helps.
Are you previously got the Ct of B actin in these RNA extracts?
-yes- Maybe it is the PCR efficiency or cDNA degradation
-No- every single animal model has a specific Ct of housekeeping genes
Evaluation of reference genes for quantitative PCR analysis of mouse lymphocytes (10.1016/j.jim.2012.07.020)
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