I am currently trying to isolate a small number of cells (200-500 cells per sample) using FACS. I sort the cells directly into an eppendorf tube containing RLT plus lysis buffer (Qiagen) with 1% 2ME (which is supposed to help block RNases). The sorts usually take between 20-30 minutes per sample, and after the sort the tubes are placed on ice. The cells are then homogenized by vortexing for >30 seconds and the lysates are snap frozen; RNA is harvested using the kit the following day. I have included a viability dye so that I am 100% sure that the cells that are being sorted into my collection tube are alive at the moment they are sorted. However, for some reason I am getting very degraded RNA according to a bioanalyzer analysis (RIN values between 1 and 3). My question is two-fold: 1) Is it possible that this is actually a limit of detection issue for the bioanalyzer and that I actually have more intact RNA but the machine is giving false readouts for RIN because the concentration is so low? 2) If these bioanalyzer results are to be believed, is there a way to stabilize the RNA in the cells that are being sorted that still allows for FACS and RNA harvest?
Any and all suggestions are welcome.
May be you can find good protocol here..
Article Purification of high-quality RNA from a small number of fluo...
Hi James,
Firstly, you can simply add DAPI to the sorted sample, DAPI+ cells would be dead. So you sort only DAPI-neg cells. Add RNAse inhibitor to the buffers you use. On my experience it is quite a common thing to have low RINs for low cell numbers. However, I received good sequencing results even from degraded samples. To use (partially) degraded RNA for RNA-seq you shall use kits for whole transcriptome analysis. In contrast to mRNA-seq kits, which rely on PolyA-containing (and therefore intact) RNA, WTA kits use random priming for reverse transcription, which do not care much about the integrity of RNA.
Best,
Michail
Abhijeet Singh Thanks for the reference, but the basic gist of that article is to sort directly into lysis buffer, which I'm already doing. Unfortunately, I think they are still using quite a bit more cells than I am, which may be why using that technique isn't enough for me to get the same quality of RNA they have.
Michail Yekelchyk As mentioned above, I'm already using DAPI (I just said viability dye in my original post), so I'm quite certain I'm only sorting live cells. Adding RNase inhibitors is a good idea, one that I've seen floated in publications that I was considering doing. I will definitely give that a shot. As for the WTA instead of polyA selection, I wonder if you have experience doing this for what a bioanalyzer says is very degraded RNA, i.e. RIN values less than 5? I've seen papers (like this one Article Effects of RNA integrity on transcript quantification by tot...
) that confirm what you are saying- that degraded RNA can be better sequenced using the random priming of whole transcriptome analysis. However, they do still see biases in the gene expression analysis, particularly in lowly expressed genes; and more importantly to me, the RIN numbers they are using in these studies don't usually appear to be nearly as bad as the ones the bioanalyzer is spitting out for me. The unfortunate thing about using the degraded RNA from this point is that I won't know if it worked until after the entire library preparation and sequencing is done, at which point I've spent a lot of money already. If you've successfully done this with very low RIN values, I'd be interested to know if you saw any biases or problems in the sequencing data.
So I was doing libraries from approx 1ng of degraded RNA, which was isolated from roughly 30-50k of stem cells, which do not have much cytoplasm and therefore do not have much RNA inside. It was so degraded, that I was not seeing the 28S ribosomal peak, only the 18S. It was showing RIN around 1 - 2. However, I had treatment group and control group, so I was doing the same library prep and analysis between the groups afterwards. PCA grouping of the samples was fine and there were plenty of differentially expressed genes with FDR
James Ropa
I suggested that paper in regard to the RNA extraction protocol from a low quantity of cells. But as you mentioned, your method is better than them, so you may please ignore that article suggested by me.
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