It's probably better to add the same amout of RNA for the RT. My guess is that you might have big differences in RT yeld depending on the amount of starting material, which might impact on your final quantification.

I completely agree with Olivier and Wilco, even when your lowest amount of total RNA will be for example 10 ng you will get enough cDNA to make a couple of tests for genes. In case you are using highly different amounts of starting material your cDNA synthesis efficiency will be different and therefore making your result after normalization just to the housekeeping gene inefficient.

Thank you for the answers. I had actually did add differing amounts the first time but they were within 10-20ng of each other. I also wanted to ask about the use of HK genes. Now if I am normalizing to total RNA and an HK gene, is it sufficient to use one HK gene that has been previously validated in my control and intervention groups or must I use two or more?

in MIQE (minimum information for qPCR experiment) papers the authors suggest to use more than one HK or control gene.the control genes should be check using genorm for instance. once validated in your experiment you can use them to normalize your data

you should now than a normalisation gene is not universal but is dependent of your experimental conditions

Hi, to use the same amount of RNA is one thing but the other thing is: Does the RNA has the same quality??? The RIN (RNA Integrity Number) value gives you the information about the quality. Here is a paper: The RIN: an RNA integrity number for assigning integrity values to RNA measurements. Andreas Schroeder, Odilo Mueller, Susanne Stocker, Ruediger Salowsky, Michael Leiber, Marcus Gassmann, Samar Lightfoot, Wolfram Menzel, Martin Granzow, and Thomas Ragg. BMC Mol Biol. 2006; 7: 3.