RNA extraction from small number of cells - (Mar/03/2006 )
I'm planning to do real-time rt-pcr for the first time and I have several samples with just about 500 cells to extract RNA from. Could you please give some advice on how should I perform RNA isolation on these cells? I was thinking of using Qiagen Microkit, does anyone has experience with this kit?
Thanks a lot
I've only had experience with the Qiagen MidiKit but found that this is a very easy kit to use. The protocol for the minikit looks pretty much the same (give this a good read cause there's a lot of good info in these handbooks).
I recommend adding the RLT (with bMe) directly in the wells and scraping the cells off the bottom of the well (although this is with far greater cell nos so maybe check that one out), we use the plunger from the inside of a sterile, RNase-free, 1ml syringe to scrape the wells, then we pipette this lysate into qiashredder columns which you have to buy separately. We tried using the 20 gauge needle technique they also suggest but found that using the qiashredders gave far greater yield.
The other thing I should say is that it's a good idea to extract the RNA on the same day as you add RLT to the wells. Due to time restrictions previously we tried adding the RLT to the wells and freezing them, then thawing them and doing the RNA extraction the next day but this greatly reduced the yield (probably due to the RNA being degraded)
(I have to add though that a lot of people don't like to use the kits and prefer the old fashion methods (trizol), which do tend to give better yields but the RNA is usually less pure. I just prefer quick, easy and less smelly methods!!!)
Hope that helps
Sorry...change all the midi's to mini's and all the mini's to micro's in my last post!!!!
Thank you Sarah for your help
I am using isolated cells by micromanipulation (not from cell culture) so that's the reason for the little amount of cells. I am going to try the Microkit, it seems more appropriate for this; we have also seen that qiashredder works well in these cases.