How to obtain RNA through laser microdissection on paraffin-embeded sections?

From:yichao wu

Dear All,

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!


Yichao WU

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.

It is a well known fact that frozen sections will NEVER have as good a
morphology as formalin fixed paraffin embedded tissue - frozens are not
fixed with NBF and then processed through solvents into paraffin, and that
is why frozen sections have poorer morphology. Having done hundreds of
frozen sections, I can confirm the frozen sections are not ideal for
morphology when compared to paraffin sections.

This is the easiest way to find glomeruli and we did it faithfully to insure they were in the section.

Rapid Hematoxylin stain on frozen section:
Fix first frozen section with 95% ethanol, you can also use neutral
buffered formalin for 1 minute.
Rinse with distilled water
Stain with hematoxylin for 1 minutes
Rinse with tap water
Blue with lithium carbonate or some bluing solution
Rinse with tap water
Mount a coverslip
Examine for glomeruli with microscope. If NONE are found, cut slightly
deeper into the block and repeat staining. When you find a glomeruli,
proceed with frozen sections, serial, for immunohistochemistry. You CAN do LCM on a DRY hematoxylin stained section. This is the way to guarantee you have glomeruli. We asked the physician for TWO biopsy cores in order to do everything.

>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).

You are probably NOT preserving the RNA, LCM methods are extremely picky and very clean to get rid of RNAse. You need to review how that is done and start asking questions. I know a lady who is an expert in LCM
and she does not handle the tissue like you are doing it. FOR LCM you must not contaminate the tissue with anything that may have RNAse or prevent that from happening. I have seen LCM done on paraffin embedded sections - as long as YOU do no destroy the RNA from the start and use the correct fixative.

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 snap frozen, cut serially, fixed with 4C acetone for 10 seconds,
air dried and immunofluorescence staining for IgG, IgM, IgA, kappa, lambda, complement, albumin, etc. This is for IFA analysis, if we wanted, we could ave done immunohistochemistry on an adjacent slide, not coverslipped it and done LCM. To do the latter, it takes special handling of the frozen section to maintain RNA. I would be collecting sections for LCM far differently than for initial routing immunofluorescent or IHC staining. 

1 piece is fixed in gluteraldehyde for EM for TEM analysis. 

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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