5 replies to this topic

[Science Badger]

Hi to everyone on the Bioforum,

I am going to be doing a qPCR of some cDNA samples soon and I have just finished gathering the RNA ready to make the cDNA. I understand from speaking to others in the lab to ensure I have the same starting template amount for the reaction in each of the samples. We have access to a speed-vac to concetrate the samples down, but I wonder if anyone could help me with the calculation I would do to make the total RNA equal in all samples once I have the nucleic acid pellet and come go resuspend in rnase-free water?

Do I take the quanitity I have measured ( e.g. 100 ng/ul, 200 ng/ul etc. ), multiply it by the total volume I have of the sample and then use the C1V1 equation to work out the rest? If anyone can give me a worked example of this I would be very grateful!

Thanks,

PhD lab newbie

[biocrazy]

e.g

Required volume of RNA to get 2 ug = 2 /the measured conc of RNA (in 1 ul)

[Tawny Owl]

Hi,

I've got a query relating to this topic, too. I remember in my masters project I was told to prepare my RNA to contain 1.5 ug in every sample I had. I was given the following formula from my supervisor:

volume of water to add (μl) = (final volume x (original RNA conc measured (ng) x total volume you had (ul)) ÷ 1500 ng (RNA conc needed)

eg.

((37 ul x 142 ng) x 8 ul ) / 1500 ng = 28.02 ul


Using biocrazy's formula example, 142 ng of RNA and target 1500 ng for instance, doesn't give the same answer  . Is the formula I was given wrong? Is there a standard way of calculating the volume of water to resuspend the pellet in?

Tawny Owl  

[bob1]

Not for the pellet itself, unless you know the mass of the pellet, however you can resuspend in a minimal volume and use the dilution formula C1xV1 =C2xV2, where C=concentration and V = volume, 1 and 2 are initial and final  respectively.

If you know the mass, then C=N/V works fine for mass per volume.

However, to answer the OP's question, the concentration of your RNA does not matter, just take the same amount in ng (not volume!) into the cDNA reaction for each sample.

[Tawny Owl]

Thanks for that, bob1. So without knowing the mass of the pellet, in the example I used, could I resuspend the pellet in 28 ul dH2O and this would mean 8 ul has 1500 ng of the RNA in it? Or have I got my wires crossed on this? Is this the same thing you were trying to do, Science Badger?

Edited by Tawny Owl, 17 November 2011 - 07:24 AM.

[bob1]

You would reusupend the pellet and then check the concentration (by spectrophotometry/nanodrop or similar) and RNA integrity (on a gel or bioanalyzer) and then take the required volume to make 1500 ng.  You will not know how much volume you need unless you know the concentration of the RNA.  Each extraction you perform will vary in the exact concentration, due to minor differences in cell numbers, handling differences etc., so you can't just rely on the pellet being the same for each sample.