How to purify RNA from a handful number of cells? - (Apr/23/2012 )
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!
I would try the types of RNA extraction kits that are being used for LCM work as these are also on a small number of cells.
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.
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!
Rule 1: Keep it simple.
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.
We used to purify RNA from single cells. From 20-30 cells the quality and purity should be very good (reproducibility is always an issue here). A related protocol can be found here:
Thanks Rsm! If I understood the paper correctly, your suggestion is not to purify the RNA but to do retrotranscription directly?
Thank you again for your reply.