about laser microdissection on paraffin-embeded sections

Dear Dr. Farsinejad,

I don't why the email I sent to you last time was returned to me. But I would like to try again.Thank you for your suggestions.I would like to know how long and how concentration of proteinase K you would use to incubate formalin-fixed paraffin-embeded sections.And at what temperature?

Thank you very much!

Yichao WU

Jinling Hospital

Nanjing China

>From: "Nima Farsinejad"

>To: yichaowu@hotmail.com

>Subject: Re: laser microdissection on frozen sections and paraffin-embeded sections

>Date: Mon, 16 Jun 2003 10:30:24 -0400

>Hi Yichao,

>The problem with extracting RNA from paraffin embedded tissue is a) Fixation,

>which crosslinks much of the genomic material (but it can be overcome by

>proteinase K digestion) and b) processing of the tissue, which can degrade the

>RNA. I have many times tried this and every time it resulted in fractioned

>RNA. Of course our goal was microarrays and not RT-PCR, so I we haven't gone

>that route yet. Last thing I remember about working on that project is that we

>were able to get lots of RNA for RT-PCR from frozen sections, but it seems that

>you have overcome this problem so I won't bore you with the details.

>Sincerely,

>Nima Farsinejad

>Lead Research Specialist

>Division of Pulmonary- Center for Translational Research

>Emory University, School of Medicine

>Atlanta, Georgia

>Phone: 404.712.2547

>Fax: 404.712.2974

>yichaowu@hotmail.com writes:

> >Another question is,since the frozen sections are not as good as

> >paraffin-embeded ones in morphology.I wonder who have successfully acquired

> >RNA for RT-PCR from laser microdissected tissue on paraffin sections?I

> >have tried and tried and failed.The kits I used are "Qiagen Rneasy Mini kit"(I

> >have no "Micro kit" at hand) and one from arctrus and one from Gentra called

> >puroscript.And after Dnase digestion,it seems that I have not acquired any RNA

> >from paraffin embeded sections.The fixation is acetone-methanol(1:1).I wonder

> >if such fixation would destroy RNA? And I wonder if heating sections after

> >paraffin embeding is destroyable? How are other procedures should be followed

> >for microdissection on paraffin embed sections? Pls help me out [

> >http://graphics.hotmail.com/emsad.gif ]?Thank you very much!