Dear Rajeshwar,

Let me ask you some questions:

1. what does the nanodrop measure exactly?

2. what is in the reverse transcription reaction? I assume you mean the RT step when you say "after PCR"!

3. why would you need a cDNA concentration for the PCR?

If think about/research these questions, you will arrive at the answer to the problem. If you can't figure it out on your own, I will help you, but take this chance to learn about what you are doing in more detail!

Just always start with the same input of RNA for your cDNA reaction (check kit, but typically 250ng - 1000 ng / reaction) and use the same dilution of the cDNA (typically 1:5 to 1:10) in your qPCR reactions, and you will have great, repeatable results.

You do not need to quantify cDNA. It will give you a larger amount. You only quantify the RNA concentration. When you perform qPCR you load the wells with the cDNA based on your original RNA concentrations up to 100ng. For example, if you have a concentration of 500ng/uL for RNA, take 1 uL of that cDNA, with 500ng/uL RNA, dilute it with 9uL of water to make a 1:10 dilution of that cDNA sample. If you have a 250ng/uL RNA sample, you can take 4uL of the sample and dilute with 20uL of water to give a 1:5 dilution. It is all based on the initial RNA concentration.

Dear Rajeshwar, at this point, you already have enough good guidance. But please just let me add one more point. After your RT, you don't need to quantify your cDNA again, you only consider the concentration of the original genetic material that you extracted. If you started your amplification with PCR, then you would have needed to quantify your DNA, but in your case you start your amplification with an RT, so you only quantify your RNA, and make the same dilution with all your cDNA.

Good luck for your manipulations.

You don't need to quantify the cDNA . Quantification is needed only for your RNA concentration.