cDNA - (Jan/23/2008 )
Hi!
Do anybody could tell me how much cDNA (mg/ml) max and min can I use for running a RT-PCR?
how much RNA are you using for the reverse transcription? i'd recommend using the one-step kit from invitrogen, the results you can get look better than making cDNA first and then the PCR since you go directly from RNA to PCR product.
it depends on your purpose.....for real time PCR i start with 1ug total RNA to make cDNA.
i use one-step kit from Roche
As said before, it depend on your experimental set-up, if you're using conventional RT-PCR or qPCR, the origin of the cDNA, the magnitude of expression of your target genes, if you need to get a quantitative result, etc. I would say than anything above 1ug is wasting your RNA. actually, it's possible to do RT-PCR from RNA from a single cell... (so they say, although I haven't done it myself!)
The best way of finding out what's the best range for your experiment in particular, I would do a test experiment with some spare RNA (if you're samples are not precious!), doing an RT with a range of concentrations (let's say, 200ng to 2ug, for example), and then PCR, and quantify the range of concentrations where the reactions is linear...
Regarding one-step RT-PCR... well, it might be OK if you have very few genes to measure, or tones of money and RNA!
the protocol mentions it can amplify from 1 pg, never done it myself either, but the lab assistant did it once with very few RNA and it worked well, however it is not appropriate to perform qRTPCR. I used up to 500 ng/ul of total RNA, when i used 1 ug/ul to make cDNA the pcr was oversaturated very quickly. try 500 ng/ul, should work fine.