RNALater

Hello everyone:

 

Here is a summary of the responses regarding reanalyzing cells sorted
into RNALater.

 

Again, my sincere thanks for your responses.

 

Best regards,

 

-Rich

 

 

 

Do not	attempt to run your samples after RNA later treatment.	There
are no longer intact cells and the sample consistency is not good for
keeping a clean clog free nozzle.

I have had great success sorting, sampling purity from sorted tube, then
spinning down to correct volume, and finally adding RNAlater.  Let me
know if you have any questions.

 

I used to use RNAlater for tissue and cell preservation.  It is
difficult to remove cells from RNAlater, as it is viscous.  You have to
dilute it with PBS in order to spin out the cells, and even then the
recovery is not 100%.

 

RNAlater precipitates proteins, so it may affect ability of antibodies
to bind surface proteins.

 

I did, and I was amazed to see the cells looked pretty good. FSC/SSC is
affected, but to a level you could live with. Markers are pretty normal,
although much of this is probably related to the actual markers used. In
our case, (although I am not absolutely sure), it must	have been
something like CD3, 4, 8, 27, 45RA ... Still, I wouldn't base a sort
purity estimate on re-analysis from RNAlater. You would be able to
"interpret" to conclude that there were no "gross abnormalities".

We have noticed that when sorting into PBS/BSA (for RNA extraction; we
prefer) CD45RA is lower during re-analysis. It must be a somewhat
fragile labeling; several hours in culture media will bring back up the
CD45RA expression level (or better "labeling intensity"). So, in this
case also, estimation of sort purity is kind of compromised.

 

Cells crumple up really well in RNA Later and the stuff is very
viscous... Good luck.

 

I tried twice with cells that had already been permeablilzed for
intracellular staining.  Both times I ended up with few if any intact
cells to check for purity.  Since then, I've always told people to sort
into buffered saline + serum and transfer the cells later into the RNA
prep buffer.  The time saving feature of sorting into the prep buffer
just wasn't worth it if you didn't know the purity of the sort.  When
someone insists, I will usually do a very short sort into saline+serum,
use that to check purity, and then put the collection tube with RNA prep
buffer in place for the remainder of the sort.	Hope that helps.