Pooling RNA/precipitation/RNA cleanup - advise needed (Aug/06/2009 )
Hi i extracted my whole blood rna using tri-reagent method, but not enough rna amount for my microarray. i want to pool my rna from aliquots and precipitate them. Howeve, i do not know how to do this as this will be my first time deal with this prob.
1. does the precipitation after pooling of rna can help to improve yield and purity?
2. what are the recommended protocols?
3. how bout the rna cleanup using Qiamp RNA kit, does it help? or should i try the precipitation first?
Really thank you with your advise. Thanks
1= yes it will improve the purity, but your recovery can vary between 50 and 90%. Normally the more RNA you start with the higher the yield (%).
If you have very low concnetrations of RNA you can add a carrier like glycogen (if this doesn't interfer with your downstream application.
2 = just google on RNA precipitation. I say this because there are many protocols and everybody has his own favourite. I just use 1/10 ammonium actetate + 2.5x ethanol 30min -80°C, 30min 14000rpm spin with or without glycogen (just like for DNA)
3 = never tried it, be aware that the lysis buffer is also the binding buffer for the columns