It is good practice to equalise the concentration of RNA in your RT reaction but it cannot be used to replace a housekeeping gene. As both the RT and PCR steps are enzyme dependent, the presence of inhibitors in the reaction can vastly change the results. Quantifying total cDNA and normalising its concentration in the PCR does not indicate the presence of inhibitors. As the PCR reaction in exponential, even a small inhibition can lead to huge changes later on. One advantage of equalising RNA concentration in your RT reaction is that with time you should know at which Ct your housekeeper should arrive, if in most reactions its 13-15 and then in one sample its 20, you know you have some problem with that sample.

Thanks Martin for your advices. But maybe I have not made myself clear. Briefly, in previous articles referees have recommend us to quantify cDNA amount used as template in our qPCR reaction because when comparing transcript levels from different genders, or even when comparign same gender oocytic transcript levels during seasonality process, we are not able to find a good reference gene. This is a problem that in our team has been dicussed for long, and we agreed to go on measuring cDNA qPCR template. (we do not have one step qPCR system). Up to now we equalized RNA previous to retrotranscription (RT) process, and after that, we diluted cDNA assuming the same efficency in all the samples. This assumption is not correct, and that´s why we used houspeekers. But they do not work at all for certain comparisons, even trying different common housekeepers. In the scientific community the use of houskeepers for certain comparisons is

being strongly discussed, and some authors are more in favour to exactly quantify the template and normalize CTs against it.

Of course, I undestand your point, and it is true that you can identify problems as you have described looking to the CTs obtained for a housekeper. However in our case we normally run more than one target gene in the same sample set, and thus we can check if enzimatic inhibition has occurred looking to the CTs obtained for the target genes across different samples in the same way that you have explained.

So, moving to our doubt and taking into account explained above, do you have any suggestions?

Thank you very much once again,