I am working with low (and expensive) amounts of tissue. Following a TRIzol extraction, I have got poor quality RNA and a low amount. I think that the main issue would be following the isopropanol step and subsequent ethanol wash steps. Since the RNA precipitate is commonly invisible, I am probably being too conservative in removing excess ethanol and therefore leaving contaminants. I have tried adding sodium acetate to make the precipitate more visible, but I don't notice much of a difference. Is there anything I can do to improve the amount and quality of RNA extracted?
Dear Duncan
The nature of the tissue that You use can be very important. Not knowing this, I can just suggest to play with the each step of the extraction, namely: tissue grinding and homogenization (probably the most problematic step in Your case, so make sute that it is done very vert well), deproteinozation (if this is the issue, try to use proteinase), precipitation (if You use alcohol - try cool it down to -20C, sometimes it can help, if alcohol is not a must-reagent to use in Your method, try to precipitate Your RNA with LiCl), and cleaning (play with EtOH%, maybe, if You use 70% try 80% and vice versa)
Try using glycogen to help maximize your yield (Trizol protocol will say when and where) and for purity, you will have to clean up the RNA following the Trizol extraction, which is common. Try the Qiagen MicroRNeasy for a column cleanup, or clean on the bench by bringing the RNA to 500ul, adding sodium acetate 50ul, isopropanol 500ul, 20minute RT incubation, then spin and wash.
I'd recommend troubleshooting with a small amount of less expensive tissue before proceeding
Thank you Albert and Carmen, I will use your suggestions in my next extraction!
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