[Histonet] RE: EDTA and DNA damage, EDTA decalcification without fixation

[gayle callis]

You wrote: 

 

Does anyone know whether EDTA destroys DNA during decalcifying process and
whether  it would be better to fix the sample before adding EDTA? 

My samples are natural coral cubes of 3mm. It is carbonate calcium. The
coral cubes are seeded with mesenchymal stem cells. I need to extract DNA in
order to evaluate cell  proliferation. 

 

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EDTA is the recommended decalcification per many publications on the subject
especially  for ISH.  You need to get this publication from J Histochem
Cytochem 47(5)703-309, 1999 as it may direct you to a protocol for what you
are doing. 

 

Effect of Bone Decalcification Procedures on DNA In Situ Hybridization and
Comparative Genomic Hybridization: EDTA Is Highly Preferable to a Routinely
Used Acid Decalcifier

Janneke C. Alers, Pieter-Jaap Krijtenburg, Kees J. Vissers, and Herman van
Dekken

 

 

Also, bone does NOT have to be fixed prior to decalcification with EDTA.  I 

 

These publications used fresh calcified bone, and decalcified with EDTA
using some stringent methods for performing frozen sections.  

 

J Histochem & Cytochem 51(1):5-14, 2003 

http://www.jhc.org

Simultaneous Detection of EGFP and Cell Surface Markers

by Fluorescence Microscopy in Lymphoid Tissues

Kim L. Kusser and Troy D. Randall

 

J Histochem Cytochem

S. Mori et al, 36(1)111-117, 1988

 

There are a  large number of publications about DNA and RNA and the effects
of EDTA and other decalcifiers on the nucleic acids.  I did a search and
brought up no less than 17 articles although did not dig through all of
them.  I presume you are making sections prior to extraction of DNA from
EDTA treated coral?  Laser Capture microdissection??    

 

Good luck you your project. 

 

Gayle Callis

HTL/HT/MT(ASCP)