How to purify RNA from a handful number of cells?
Posted 23 April 2012 - 06:28 AM
In our lab, we are trying to separate cells found in urine by cell sorting. I would like to purify RNA from these cells. The problem is that we might have 20-30 cells all in all and I guess the conventional extraction methods just as Trizol or any kit fall too big for such a small volume. Any idea on how to overcome this problem?
Thanks in advance!
Posted 23 April 2012 - 10:33 AM
Eg. Ambion's RNAqueous. I would also expect that you will require a post-extraction amplification of whatever you are able to recover for these cells as well though.
Posted 24 April 2012 - 02:37 AM
I checked RNAqueous information out and I don´t think it is suitable for my needs. I work with just a few cells!
But thanks anyway!
Posted 24 April 2012 - 06:21 AM
Too many manipulations and you're going to lose your sample.
So, though I don't have a protocol to hand, I'd think about what you want to do:
First concentrate your sample - Millipore Amicon or Centricon units?
Protect RNA - add a chelating agent to mop up MgCl2 which is a co-factor for nucleases - something like Chelex-100 ?
Then get rid of everything you don't want - Proteinase K digestion and DNase digestion?
Final Amicon of sample to get rid of cell debris and concentrate sample?
Keep it as simple and the manipulations as basic as possible.
Posted 24 April 2012 - 10:47 PM
Posted 24 April 2012 - 11:47 PM
Thank you again for your reply.