thanks for your quick reply.
We use the same staining protocol as you do. We have modified it by addinng RNA-later for 5 secs and short washing in DEPC-PBS 1% before starting with rehydrating with etoh. We have not used xylene, but nonetheless our protocoll worked perfect.
Curiously, when I started with the staining, we had the problems described above.
So I tried to exclude a lot:
RNA-later yes/no: no difference
PBS yes/no: no difference
air drying: no difference
xylene yes/no: no difference
keeping slides cool/warm: no difference
methanol before etoh: no difference
discarding all reagents and replacing new reagents: no difference.
Interestingly, suddenly our problems with bad staining stopped (whyever?!) and everything went well for 1 year.
Now, our staining quality has decreased suddenly again. Even samples, which worked perfect in the beginnning, have acquired this "dirt" staining now.
I have no explanation, especially, since we excluded every new factor.
I keep on trying - maybe the problem will resolve by itself once again
Does anyone know, whehter this staining problem has a negative effect on rna-quality?