If I switched the final volume of my qPCR reactions from 50uL to 25uL, will it interfere in my analyzes of my biological replicates, once I already started the analyzes of some replicates with 50 uL?
Can I compare biological replicates in qPCR with different volumes?
http://www.gene-quantification.com/dorak-book-real-time-pcr-2006.pdf
It's not advisable, as reaction dynamics are not necessarily linear. The analysis of biological replicates using qPCR is relative, so if you adjust the concentration of template (by half in your case), it is more than likely that the Cq values for these samples will be higher than if you assess them at 50uL. However, if you run all samples at either 25 or 50uL (not a combination of both), you should be fine.
Dear Amanda
I would try to mantain the amount of sample in the lower volume. You may reanalyse in 25 uL one of the samples already tested in 50 uL, keeping the amount of template. Do 5 replicates and estimate the coeficient of variation. Then you can better decide.
Good luck.
Dear all, based on the above question and all of the answers, I believe that if the amount of starting template (cDNA) is same, the results should be same. After all, the volumes can be increased by merely adding RNAse free water but that has nothing to do with the amplification process. Thus, if I add 10ng of cDNA+15uL of SYBR green and water (suppose) in one well, and 10ng of cDNA+40uL of SYBR Green and water in the other well, the amplification in both cases starts with the same number of cNDA strands and hence, the results should be identical. Please let me know if I missed any point and came to an incorrect conclusion.
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