I'm doing qPCR on four copies of ATPase alpha. The expression level varies a lot and the Ct values at two cDNA concentrations are as below:
cDNA Copy 1 Copy 2 Copy 3 Copy 4
~1ng 28.9 18.9 8.9 29.7
~0.1ng 30.9 22.0 12.0 32.5
Usually people adjust the template concentration to make Ct around 20-35, so I wonder if the ~10 Ct value of Copy 3 is not reliable.
But another thought is even if the extreme Ct values are not that accurate, it's still an obvious indicator that the expression of these copies are very different. Am I right?
Thanks!
I believe CT values between 23 and 35 can be chosen to build a curve to monitor qPCR efficiency.
Napur is right, a data point at these very low Ct values should be tagged as possibly inaccurate. It really depends on the goal of your experiment and what method you are using in order to gauge the expression level of ATPase. If all you are looking for is a simple binary difference, these low Ct values are of lesser concern. If you are looking to track many different treatments, with potentially small differences in expression level, and simultaneously high expression levels (low Ct values), then these will make it difficult to determine those potentially minute differences. From what you say, it appears you really are looking for the larger differences, so the fact that this copy has a much greater expression level, may not be a hindrance. But you will have to justify that point (depending on where you publish), which may require that you dilute your cDNA about 3-4 more times, and get a reading in the more accurate Ct range for that copy. You can then confirm the accuracy of the low Ct value with some simple calculations.
If you do not have reliable Ct Value, you can not conclude there is high copy number eventhough that is correct. this may be fake amplification as well. Another thing I did not get is why your copy 4 has higher Ct values than C1-C3.
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