Reverse transcription question - Volume for RT reaction (Jan/29/2010 )

I know that standardly we use a 20 µl reaction volume for second strand synthesis of total RNA using 1-2 µg RNA but does anyone routinely use higher reaction volumes? Do you still get adequate results??

I am asking because I have very limited RNA in high volume and have way too many samples to concentrate. If possible, I would like to use a 50 µl reaction volume using 1 µg RNA. Just need someone else to say "it'll work" and then I'll feel more confident :-)

AussieUSA.

AussieUSA on Jan 29 2010, 02:30 PM said:

So, my concern with the increase in reaction volume is the accessability of the enzymes and dNTP to the RNA template- so, if you have a larger volume, the reagents might not be able to 'find' each other with all the extra fluid floating around. It might work, but probably not with good efficiency is my guess. Also, you might not get the proper salt/buffer amounts if you use increased volumes, and then the reaction might also be off. My advice would be to keep the 20 µl reaction volume and just put in 0.5 of your template. I've definitely synthesized second strand amounts in much less quantity than that and gotten good reaction outcomes.

If the volume of each component of the reaction is increased proportionately, I see no reason it shouldn't work for an RT. I'd be hesitant to do this in standard or real-time PCR because larger volumes will heat up/cool down more slowly in the short cycle times.

gfischer on Feb 1 2010, 06:42 PM said:

Hi,
well, increasing the amount of reagents might be some some question of money, besides, a raction volume of 50 µl has indeed some volume issue. But I would still use a cycler for the procedure, perhaps including some adaption of the time steps. Cyclers are much better than any heating block as you have the heated lid and really exact temp control.