I need to run a series of genetic expression assays using RT-qPCR.
I will use housekeeping gene to normalize the output of the genes of interest.
Shall I normalize the experiment in some additional ways? For instance, the amount of RNA that goes into the reverse-transcriptase step or the ng of cDNA that goes into the qPCR reaction?
Thank you
Always use the same amount of input RNA whenever possible: why would you _not_ do this? Otherwise you're simply introducing additional variation for no reason.
Spec your RNA, because you need to do this anyway to establish
1) how much RNA you have (if indeed any)
2) how clean it is
Assuming you have decent yields (500+ ng.ul-1) and good purity (260:230 ratios of 1.7+, ideally 2.0+), then use the same total amount of RNA for each cDNA reaction.
If you can, aim for the upper end of what the kit can handle, since...why not?
If you have one or two samples that won't permit this (too low [RNA]) then for those specific samples, just use as much as you can, and make a note of it.
Much better to have one or two samples you know are low [input], and all the rest at high [input], than to lower everything else to accommodate just those two.
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