Normalising RNA concentrations - Help (Jun/01/2010 )
So I am doing some cDNA synthesis this week. I have several RNA samples which I need to reverse transcribe to cDNA. I am going to use the cDNA in qPCR to look for differences in gene expression between the samples. I have to make sure that my samples all contain the same concen. of RNA before I start the cDNA synthesis. Currently they are all very different:
86 ng/ul, 120 ng/ul etc.
I want about 2 ng/ul for the cDNA synthesis, so have to dilute them but how do I calculate how much water to add to get them all a 2 ng/ul.
Can I use C1V1=C2V2? I am so out of my depth with this as have barely done any molecular biology, can anybody explain this to me in simple terms ??
C1 (V1) / (V1 +V2) = 2 ng/ul;
where C1 is the current conc. in ng/ul, V1 is the vol you want to aliquot from the stock, (V1 + V2) is the final volume, V2 is the vol of sterile water you need to add and 2 ng/ul is the desired concentration.
E.g. Your stock conc is 120 ng/ul, and you want to dilute 1 ul of the cDNA to final concentration 2 ng/ul.
120 (1) / (1 + V2) = 2
120 = 2 + 2*V2
V2 = 59 ul
In other word, it's a 60 x dilution of your stock
why do you want to dilute the RNA before the cDNA reaction that much? usually you take for instance 500ng up to 2µg RNA for one cDNA sythesis reaction (20µl) and then you dilute this to the desired amount you want to use in real time PCR. Keep in mind that the ingredients of the RT reaction are inhibiting the PCR so you have to dilute this out after the RT.
The reason I am diluting so much is that I am using the Roche Transcriptor high Fidelity cDNA synthesis kit, the protocol for this specifies that I use 1 ng - 4 ug of total RNA in each cDNA reaction. The protocol says that the cDNA can then be used in PCR directly and suggests using 1-5ul of the cDNA reaction in a 20 or 50 ul final PCR product. I have never done this before so was just following the Roche protocol to the letter. Am I about to make another mistake???
Sorry I just re-read that - 1 ng - 4 ug !!!! of course I don't need to dilute I think I had thought it said 1 ng - 4 ng - sorry that was a very stupid mistake to make!!!