[Histonet] Re: LCM & archival FFPE tissue (Margaryan, Naira)
Suresch, Donna L.
donna_suresch <@t> merck.com
Wed Feb 23 10:57:30 CST 2011
I have used archival FFPE slides that had used antigen retrieval methods for LCM and the yields seemed to be good. The RNA/DNA yields obviously depend on how many cells you are collecting. We routinely used 8um sections for the LCM to increase yields. If you are interested in collecting a population that is very limited in your samples (i.e., ER+ or PR+ in small tumors), you will need to have maybe 20 or more slides (estimate). I would collect those slides into a single microcentrifuge tube (insert LCM cap and invert it to get cells into buffer) using lysing buffer, heat at 42C for 30 min, spin at 2000x g for 1 min, decant buffer, resuspend pellet into RLT/70% ethanol, and freeze the samples at -80 degrees C until ready for PCR.
There are some excellent protocols from Quiagen online that will be helpful.
Hope this helps.
Donna L. Suresch
Merck Research Laboratories
Imaging Research - West Point
Mail Stop: WP44K Office: WP44-H129
770 Sumneytown Pike
PO Box 4
West Point, PA 19486-0004
<<Suresch, Donna L..vcf>>
Date: Tue, 22 Feb 2011 13:26:47 -0600
From: "Margaryan, Naira" <NMargaryan <@t> childrensmemorial.org>
Subject: [Histonet] LCM & archival FFPE tissue
To: "histonet <@t> lists.utsouthwestern.edu"
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I have a couple questions I would like to throw out to the experts:
1) 1) Is it possible to extract RNA and DNA for gene expression purpose after usual IHC procedure on 4um sections of archival FFPE breast tumor tissue (If tissue was in citrate buffer, pH 6.0; several blocking steps; one hour in primary Ab (less then one hour will not work); with immunoperoxidase-DAB detection follow the Hematoxylin?
2) Or I need only H&E?
2) 3) Is 4-5 um section good enough?
3) 4) How many sections need to have really good and highest yields?
4) 5) What temperature of Proteinase K is need 42Âº or 60ÂºC?
I've the LCM system from Arcturus and the Pico Pure Kit.
Thanks in advance,
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