I'm running qPCR to study gene expression within two brain regions, the prefrontal cortex (PFC) and nucleus accumbens (NAc) in rats. My goal was to input 10 ng of cDNA per well for each region. For PFC I produced 900ng of cDNA from RNA, with an original concentration of ~42.86 ng/µL of the cDNA sysnthesis reaction, I diluted correctly by taking 3.85 µL of stock into 33 µL total (yielding ~5 ng/µL and thus 10 ng per 2 µL well). However, NAc stock was ~34.29 ng/µL (720ng produced in a 21 ul reaction volume), and the correct dilution should have been ~4.81 µL into 33 µL to achieve the same final concentration. Instead, I mistakenly used 3.85 µL for NAc, which resulted in roughly 4 ng/µL final concentration and about 8 ng per well, which also worked without a problem for a first plate.

Originally I didn't plan to do direct comparisons between the two brain regions. In this case, having a different cDNA input for each region would be acceptable I assume? Since within the regions as long as the cDNA input is the same the relative quantification will work.

But let's say I would like to compare results of both regions:

Is it acceptable to compare gene expression between PFC and NAc if one region was run with 10 ng per well and the other with 8 ng per well, assuming proper normalization with reference genes within each region.

Or would you recommend re-running the NAc plate to match the input of the PFC?

Any insights or experiences with similar situations would be greatly appreciated.