Amount of RNA for cDNA - (Jun/04/2015 )

Hi,

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.

Thanks

if you have 19 ng/ul when you start and then take 2 ul you have 48 ng, which is higher than the 2 pg minimum for the kit, so it should be fine

bob1 on Fri Jun 5 02:33:49 2015 said:

if you have 19 ng/ul when you start and then take 2 ul you have 48 ng, which is higher than the 2 pg minimum for the kit, so it should be fine

Relieved.

Just a correction it should be 38 ng for 2ul.

Correct... shows I should proof my posts before I send them.

Hello Newbie,

Considering you would use your cDNA for Q-PCR, should you not convert a uniform volume of RNA into cDNA e.g., converting 50ng of RNA for all samples.

If you take in 38ng, then all samples you are going to test should be final concentration of 38ng!!!