I have isolated RNA from as low as about 5000 sorted cells. You can use Qiegen's RNeasy Micro Kit if the Mini kit elution volume is still too large..

thanks for reply,

I also have to downscale it because I want to sort for the small LT-HSCs from mice. I read that there is an option to sort directly into the lysis buffer. When you sort directly into the first lysis buffer in the KIT, how long can you keep the cells there (because I want to sort more than one fraction of course and this needs time)?

Christian, I would suggest you to sort into Trizol reagent (the time is not critical here) and purify your RNA right from Trizol lysate using Direct-zol RNA kit from Zymo Research (Epigenetics). I have a great experience with their chemistry.

I used the RNeasy Micro Kit from Qiagen - I sorted different cell number (500, 1000, 5000, 10000 c-Kit+ cells) into lysis buffer RLT. Then I proceeded according to the protocol. I eluted RNA in 14 µl nuclease free water from the KIT. When I measured the RNA on Nanodrop, I got of course very little amounts (this I expected) but I could also see a huge peak at 230 nm - 280/260 around 1,7 and 260/230 around 0,7. Is this normal if you have so little amount of cells/RNA or is the quality of the isolated RNA just bad (contaminated)? I tried this now 3 times and I had always the same problem. Can anybody help??

Thanks again

FIrst of all, there is always very little RNA from these small populations even if you get reasonable number of cells. The risk of sorting directly into lysis buffer is that you might get contamination from other populations. I often check the purity before I sort them into lysis buffer (mainly Trizol). You will need as many cells as you can possibly get.

Thanks Milon. I would have still some questions more:

(1) when i would like to sort lets say 5000 LSK cells into trizol, which volume of trizol would you use?

(2) are you combining the trizol extraction protocol with some other KIT - some people use trizol + Qiagen RNeasy Kit

Thanks again.

I normally sort them in 500uL of Trizol with the cells go directly into it so that you won't lose the cells, and I use chloroform/isopropanol to purify it. I am sure that the kit would work find too. Good Luck!

Hi, I would like to know if after trizol extraction you clean up RNA with RNA Clean up columns (Qiagen) or similar. Just with trizol, I get very bad ratios.

Thanks!