about laser microdissection on paraffin-embeded sections

From:yichao wu

 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.
>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!

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