Dear Menno,

I am quite interested in your quest to get good quality RNA from PFA fixed FACS sorted cells. I wonder how did you experiment go?

BW

Huseini

Yes, start reading here for example: http://www.uvm.edu/medicine/vtcancercenter/?Page=facilities_flowcytometry.html&SM=facilitiessubmenu.html

But: it is a lot of work and you need to make sure the FACS is RNase-free. This may be impossible if you only have access to a shared FACS. Anyhow, the first thing you should do is calculate if you will have enough cells after sorting ;-)

This article might be helpful: Gene expression analysis of in vivo fluorescent cells.

Khodosevich K, Inta D, Seeburg PH, Monyer H.

PLoS One. 2007 Nov 7;2(11):e1151.

The fixation with PFA may be reversible and using the right RNA extraction method you may get really good RNA. If you look at heir protocol for sorting cells from mouse tissues, it is surprising how good the quality of RNA is. There is no cell sorting involved but they do an even worse thing: they do laser capture, which is at room temperature. But their cells are completely dehydrated. Hard to say how the FACS will damage the RNA...

However, since the above paper uses tissue as a stating material, cell culture cells seem not do do so well when fixed in regard to RNA integrity: PLoS One. 2013 Sep 2;8(9):e73849. doi: 10.1371/journal.pone.0073849. eCollection 2013.Quantitation of gene expression in formaldehyde-fixed and fluorescence-activated sorted cells.Russell JN1, Clements JE, Gama L. But this paper did not use a lot of variation in fixation. there is probably a lot of improvement possible and 4% PFA (I assume this is the concentration you intend to use) may be better than their fixation. Plus the second manuscript uses a Qiagen kit and a Qiagen kit failed to give any result in the first manuscript. A lot of tinkering may be necessary...

You can try this method but the only problem is that the method has lot of steps involved which decrease your RNA yield at the end and FACS will damage your RNA. There will be more exposure to Rnases. May be you can increase your sample size so that you get a right amount of RNA at the end.  

Dear Menno,

Theoretically you can use FACS to isolate yourr desired cells for later RNA  collection. However there are lots of variable with FACS which makes it a unhealthy choice regarding that in a department several  gruops using the FACS machine, chance of RNase contamination and laso the unstability of RNA even under te best conditions.

Regards,

GCK

Thanks for the answers / advise,

I did the sorting (indeed with the FACS), and ended up with 0.25 - 4 *10^6 cells per sample. As a control I also took along fresh (unfixed) cells. I added trizol to all samples and put them in the -80 freezer. Tomorrow I will first isolate the RNA from a fixed and a control sample and I'll see how it works out. I'll let you know.

best,

Dear Menno,

I am quite interested in your quest to get good quality RNA from PFA fixed FACS sorted cells. I wonder how did you experiment go?

BW

Huseini

Dear Menno,

May I know how did your experiment went in trying to get good quality RNA from PFA-fixed FACS sorted cells?

Appreciate your sharing

Eddie

Meno, may I know how it went?

I'm also VERY interested in this experiment.

We are sorting rare cells out of a tissue sample that has been dissociated into single cells. We are using Ambion's RNAqueuoes-micro kit to produce RNA, and we sort cell by FACS directly into the kit's Lysis buffer (which has guanidine thiocyanate and beta-mercaptnol, immediately protecting the RNA). 

I would love to be able to sort fixed cells as our procedure right now is very complicated and sorting live cells makes it even harder...

Dear Meno

How did the extraction went from fixed cells??

Thank you

This might help.

http://dx.doi.org/10.1371/journal.pone.0073849

answer pls

Dear Menno, how did your extraction go. I will like to try i fit worked. Answer please

In the end the experiment was cancelled, so I don't know the answer.

best,

In the end the experiment was cancelled, so I don't know the answer.

Thanks, Claudia, for recommending my old paper. In it, we tested PFA, ethanol/acetic acid and methanol for fixation, only E/AA worked. You need to add an RNAse inhibitor into the fixative, and be extremely careful with all solutions and sorter tubings. Also, yield is low, as fixation allows RNA to leak out of the cells.

Dear all,

Did somebody finally did RNAseq after fixation and cell sorting ?

I plan to do a single cell isolation solution from tissue samples, then fixe this solution and do cell sorting by FACS (the next day) and put it in trizol for further RNA isolation.

what do you think ?

Thanks++

Dear Alexandre,

Very interested to know how your isolation went. I’m planning on fixing my cells, sort by FACS the same day, then immediately putting them in trizol for RNA isolation. Similar to what you described. Where you able to get good quality RNA?

Best,