I am analyzing the expression of eight genes including the housekeeping gene GAPDH, in a sample using two-step RT(reverse transcription)-PCR. I understand the importance of including a no RT control to eliminate the possibility of gDNA contamination. I just want to ask if a no RT control should be performed for all eight genes or is it ok to just perform it for the housekeeping gene only and assume that the sample RNA is free from gDNA. Thanks.
Need no RT control for all genes?
Posted 26 February 2014 - 10:44 AM
If you have assay that will amplify gDNA if present, you may actually check the gDNA presence. If this one RT- assay is negative for all samples, then you don't have any gDNA in them. In that case, EVERY PCR you do on those samples is cDNA only. You don't need more controls.
If you have assay that may or may not bind gDNA, some more some less, then you can't be really sure if the single one you picked is just not binding DNA enough, and some other might, so those other results will be affected. So it's important to select the right one or run all.
If you have only assays, that will NEVER bind gDNA (on exon-exon boundary, checked prior on gDNA samples), then you can't get a possitive RT-, but at the same time you can't check for gDNA contamitation, that affects also the reverse transcription, if the gDNA content is significant.
Also, in some cases, you may use RT- as a "background", even if it ends possitive, because you substract the difference. But in this case, you need RT- for every single sample and gene ran.
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