I know this question must have been answered before but i would like to confirm it again.
I am using Thermo-scientific cDNA synthesis kit and it says take 1pg - 5ug of your template for cDNA synthesis. I am having a RNA yield of 19 ng/ul (micro-dissected) brain sample i.e. the hippocampus with a total volume of 50 ul. I then treat it for DNAse. So, my total volume is not more than 55 ng/ul and i want to save some RNA for future experiments.
Now, to calculate 1 ug,
x ul = (1 ug)/(25ng/ul)
x ul = 1000/19
x = 52.63 ul
Until now i was using about 2 ul of the template to synthesis cDNA. Will it be fine with 2 ul or should i inc. the volume to 50 ul for cDNA synthesis and then proceed on with the qPCR? Although the cDNA synthesis kit gives a final volume of 20 ul after addition of the template.
Amount of RNA for cDNA