Hello

cDNA normally is not subjected to direct quantification. You can calculate the amount of input RNA incorporated into your qPCR reaction. This is done by knowing the dilutions during the whole process.

Good luck

Eduardo Alvarez Let's say you have taken 100ug of RNA in 20ul reaction volume for the cDNA synthesis then you assume that all of it is converted to cDNA giving the final concentration as 5ug/ul. The cDNA is not to be quantified because it would be very difficult to select the blank. Now accordingly you add the cDNA in your last step. For example, for 10ug cDNA, you would now add 2ul of the cDNA solution.

Eduardo Alvarez As correctly said by Ali and Vipul that cDNA is not subjected to direct quantification for their use as template in RT-qPCR. Instead, the following rule is followed:

Use exactly same quantity of starting material (RNA or mRNA) as template for cDNA synthesis and follow the similar steps for cDNA synthesis of all the samples (DNase treatment, reaction volume, temperature cycles, cDNA synthesis kit, etc.) and then for RT-qPCR use the equal volume of cDNA (of same dilutions) as template. Then you do not have to worry about the variation in cDNA concentration.

Moreover, use of internal control or reference gene(s) is suggested to normalise the expression of target gene(s), which takes care of these variations, if any.