I've seen in MIQE guidelines that calibration curves a needed when performing RT-qPCR. I'll use genomic DNA to perform these calibration curves.

My question is that I don't know the range of concentrations to test.

The genes I will check for differential expression have logFC between 2-8 (base 2 log).

50 ng/μL is the sample concentration I will have when performing the Comparative CT Experiment, but 98% is ribosomal RNA.

Please let me know you suggestions and possible reference books/papers to revise.

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    $\begingroup$ I am not sure what you're trying to do. I think you want to measure gene expression level (amount of transcript), but then why would you use DNA as your reference? What do you want to calibrate with the DNA? $\endgroup$ – yotiao Dec 5 '13 at 12:07
  • $\begingroup$ Are you doing a gene expression analysis by relative quantification of cDNA? Or are you looking for copy-numbers of a gene in genomic DNA? In either case, you need the same type of template in both the standards and samples, and that your curve must cover the whole range of Ct's in the sample set for the most trustworthy results. For cDNA, I usually get a purified and concentrated cDNA from the same cell/tissue/etc. and do 1/4 dilutions for 5 points (every dilution giving roughly +2Ct) $\endgroup$ – WesternBlöd Apr 17 '15 at 13:32

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