RNA conc importance? - (Nov/04/2008 )
I have some samples which have v.low RNS concs (not mine given to me).... so the volume of RNA needed for 1 microgram is very high. I was going to try use 0.25 microgram in my cDNA step. However in our protocol we already do a 1:5 dilution of RNA (1microgram) and primers and oligo dts (made up to 20) then 5 of this into the rt reaction. So then I would have 0.05 micogram cDNA and then the 1:10 (ie 2 microlitres) for the pcr, would mean I would have a conc of 0.0025. Is this too low for PCR? Or should my fragment still amplify?
I think it should be ok, but wanted to get others thought b4 wasting my RNA...
You can use the maximum volume of RNA in your RT reaction, do PCR without diluting the resulted cDNA, or you can concentrate your RNA by ethanol precipitation or using a speedVac. If you use less RNA for RT and still want to dilute cDNA, PCR should be fine if the expression of you genes of interest is not too low.
So it should be ok to do the RT with volumes ranging from 6 to 50 microlitres (this is with 0.5 microgram of RNA, dont have enough volume for the whole microgram) . Do I then need to change my RT rxn recipie to correct for the volume and thus concentration changes of the other reagents (dNTPS, RT enzyme etc?) at the moment we use x microlitres of RNA, 0.5microlitres oligo dt, 0.5microlitres random primers, 5microlitres rnx buffer, 1.5microlitres dNTPS. Or should I keep these the same as the amount of RNA is not changing just the volume it is in. Would you suggest to use then 5 micorlitres of this cDNA ni the PCR.
THanks for your help.
Its hard to trouble shoot and try things out when the conc is so low and I'll end up loosing the RNA... so your help is very usefull!