what's the sufficient concentration of RNA for reverse transcription? - (Aug/10/2006 )

I isolated RNA (trireagent method) and measured it in photometr, I have very different concentrations of my RNA from 30-1400 mikrograms/ml. I'd like to know what's the sufficient RNA concentration for use it in RT-PCR

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Hi

the smallest amount of RNA that worked for me was 300ng , purified with TRIZOL and using Roches Expand RT. Below that i did not get any decent results from the lightcycler PCR.

greetz

tobi

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but was it 300ng/ml?? seems very little. but very thanks for answer, help me to get overview

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so maybe You can answer me another question if You using TRIZOL, is Your RNA pellet white or colorless at the last stage of isolation?? cause once I had really nice visible white pellet and another time I had "nothing", I mean I couldn't see anything at the bottom. is that mean that I had no RNA?
thank You for help

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I've used as little as 80ng RNA per RT reaction successfully.

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I think tobikenobi was talking about 300 ng. usually it's in few microliters.

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Hey, I do viral RNA extraction, and most of the time I don’t see any pellet (nothing)!!!! And my RT-PCR works perfectly well (I never quantify). Actually, I started to add glucogen to be able to see a pellet.

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We're doing RT-PCR on 10 µl of a 50 µl eluate, when extraction started with as little as 250-500 copies of RNA and we're pretty succesfull. Not doing real time PCR, but regular RT-PCR (nested btw with specific primers).

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