|From:||yichao wu |
Since there are many archival paraffin-embedded sections in my department, we would like to do laser microdissection on this kind of sections and obtain RNA for subsequent study.But I never succeed up to now.
I have two questions,
1) It is said that RNA is unstable until it is frozen or incubated in a fixative.
Does this mean that RNA is safe when the specimen is fixed? (the Rnases are also fixed and have no activity?) From what step the RNA would be unsafe? Is the process of cutting paraffin-embeded sections dangerous for the safe of RNA?
2) I learned that fixatives include two types. Crosslinking ones like formalin would crosslink protein and RNA but would not destroy RNA. Precipitating ones like acetone would destroy RNA if the incubation time is too long.Is it right? I just realized that our technician always incubate biopsy specimens overnight in acetone-methanol fixative for convenience(She will be off-duty after two hours incubation in fixative).That may be the reason that I always don't get any RNA on such fixed paraffin sections. Am I right?
And another choice for me is sections fixed by Bourin-HgCl2.Is this kind of sections usable? I mean would RNA be maintained in this kind of paraffin sections?
Any comments are welcome.Thank you very much!
Jinling Hospital, Nanjing University, Nanjing,P.R. China
>Gayle Callis wrote,
Perfect morphology on a kidney frozen section is not possible, it is the nature of the method. You can have good morphology but not the same as paraffin, see comments below.
On the other hand, I still want to improve the frozen section protocol further for better morphology to the uppest level.I wish someday my director would like to transfer to frozen sections for both IHC and molecular analysis. Maybe the morphology would be a little worse, but we have better material for laser microdissection and in situ hybridization.
This is the easiest way to find glomeruli and we did it faithfully to insure they were in the section.
>You wrote: Therefore,on one hand,we are trying to do laser >microdissection on such acetone/methanol-fixed paraffin-embeded >sections,wishing to acquire RNA(But we tried and tried and failed).
The kidney biopsy protocol for a renal pathologist here was
1 piece is fixed with neutral buffered formalin, processed and embedded in paraffin, serial sections are cut at 2 um for H&E, Massons Trichrome, PAS-hematoxylin, Jones methenamine silver for basement membrane. These sections are for light microscopy analysis, that is what she should be looking at for diagnostic light microscopy.
1 piece is fixed in gluteraldehyde for EM for TEM analysis.