How to extract 20 bp dsRNA? - extraction from roots (Aug/01/2005 )
Is there anyone that knows how to extract dsRNA (around 20bp) from a sample of plant root? Any suggestion will be wellcome.
Thanks in advance
columns are not ok for that because the cut of is greater than 20bp. (correct me if i'm wrong...)
Tri family are ok for that. (trizol and tri reagent)
This is tough. Your best shot is probably a microcon filter with a 10,000 Da or less cutoff. Keep the flowthrough, which will have short fragments. But it will be difficult to separate these from salts or other contaminants.
Can we ask why this is part of your experimental plan?
It sounds like he/she is investigating miRNAs, or working on RNAi in some sort.
yes i am working with iRNA and i got a protocol to separate them, working with the supernatant of a simple RNA extraction,and after several steps with LiCl, ethanol and phenol-chlorophorm to wash it i can detect with a blot. But because dsRNA is not that easy to get this way, i need to process around 3 gr of roots and it is not always enough, without saying that i need to do several samples at the time. So, i was wondering if there is another way to do it. Maybe using some column. .........
I am just starting my master course, and trying to set the experiments...