We are trying to isolate high quality RNA from PFA-fixed and permeabilised cells that are then subjected to FACS sorting after antibody staining for intracellular markers. I have read that one of the most "dangerous" points in the procedure for the RNA is during antibody staining step as the cells are resuspended in an aqueous solution, increasing the likelihood of attack by endogenous RNases.
RNAlater has been used in conjunction with FACS sorting by others (Zaitoun et al. BMC Research Notes, 2010) although this was using endogenous fluorescence, not intracellular staining.
My question is: if I also incorporate RNAlater into the antibody staining step, is it likely to denature the antibody (given that the ammonium sulfate in the buffer denatures certain proteins incl. RNases)?
Thanks in advance for your advice.
Rnase inhibitors added to all water containing steps effectively preserve RNA. I routinely use them for the immune stains for laser capture microdissection. There are many of them on the market, e.g. RnaseOut from Invitrogen.
I hope it helps,
Yelena
It does. One reason why RNAlater is kept at r.t.p (mimics the body's temperature) before aliquoted in the sample is to excise the cells and permeates rapidly within as it protects any mRNA and during storage at -80 degrees Celcius for future storage.
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