Dear all,
im performing a protocol in which i circularize the cDNA and then do the amplification PCR of the libraries.
I process many samples, which i would prefer to treat identically as much as possible not to introduce any bias.
With 12 PCR cycles some samples look great, some rather overamplified.
(little note: I am aware that the overamplification can create artifacts and eventually clonal reads. But after performing the PCR i precipitate it and size select on a gel, so i dont have in my libraries any slower migrating byproduct).
With many samples performing differently, it seems that keeping the PCR conditions similar for all of them is not really an option. Lowering the cycles would make me loose the samples where i seem to have lower cDNA input.
My fear is however that varying the number of cycles for each sample will affect the quantitative interpretation of the data after sequencing (e.g. due to preferential amplification of specific products).
I am afraid to introduce an unexpected bias, which would be deleterious if i want to do quantitative comparisons.
What are your thoughts on that?
im performing a protocol in which i circularize the cDNA and then do the amplification PCR of the libraries.
I process many samples, which i would prefer to treat identically as much as possible not to introduce any bias.
With 12 PCR cycles some samples look great, some rather overamplified.
(little note: I am aware that the overamplification can create artifacts and eventually clonal reads. But after performing the PCR i precipitate it and size select on a gel, so i dont have in my libraries any slower migrating byproduct).
With many samples performing differently, it seems that keeping the PCR conditions similar for all of them is not really an option. Lowering the cycles would make me loose the samples where i seem to have lower cDNA input.
My fear is however that varying the number of cycles for each sample will affect the quantitative interpretation of the data after sequencing (e.g. due to preferential amplification of specific products).
I am afraid to introduce an unexpected bias, which would be deleterious if i want to do quantitative comparisons.
What are your thoughts on that?