Hi!
I wonder about total RNA concentration that is fixed before cDNA and qPCR. so why we do that since the delta delta ct will normalize the different in gene expression between samples ?
Because less or higher amount will disturb your final results. that is why we fix the concentration.
Hello Batool Ismail, how are you doing today?
Well, there a couple of reasons why. First, total RNA is not just mRNA, you have transporter RNA, Ribosomal RNA, microRNAs, and etc. Second, the most kit available for cDNA synthesis use standard concentrations around 1ug of RNA for cDNA synthesis, and if you are using oligoDT primers just mRNA will become cDNA, but if you are using random primers, all the RNA present in your sample will be synthesized as cDNA. Third, usually in qPCR, you are trying to access mRNA expression, so standardized the samples is required to have enough mRNA for the qPCR reaction and the CT values does not become too different between samples for both your target genes and the housekeeping controls. Well, I hope that will help you. Good luck with your research.
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