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An efficient and rapid method for DNA extraction from formalin fixed

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An efficient and rapid method for DNA extraction from formalin fixed Powered By Docstoc
					An efficient and rapid method for
DNA extraction from formalin fixed
and paraffin embedded tissues
suitable for analysis by polymerase
chain reaction (PCR).

E. Gallo, G. Di Marco, M. Stella, G. Costanzo, M.R.
Rizzuto, F. Raiata and A. G. Rizzo

Unità Operativa di Anatomia Patologica, Azienda
Ospedaliera “V. Cervello”, Palermo
Background:

• Formalin-fixed paraffin-embedded tissues are a
  valuable source of DNA for molecular studies due
  to the availability of large tissue archives in
  almost all hospital pathology departments.

• Reported in the literature there are several
  methods for DNA extraction from paraffin
  embedded tissue and most of described methods
  report an amplification success rate of 60-80%.
OBJECTIVE :
 A wide variety of methods exist for the recovery
 of DNA from formalin-fixed paraffin-embedded
 tissue sections and the technical procedures for
 DNA extraction include many steps such as
 deparaffinization in xylene and enzymatic
 digestion (or other chemical treatment coupled
 with enzyme digestion).
 In a prospective study we compared two methods
 to identify an optimal and rapid protocol for DNA
 extraction from paraffin-embedded tissue
 samples.
MATERIAL AND METHODS:

• Tissue samples:
 Nuclear DNA was extracted from forthy-five
 cases of surgically resected colorectal
 adenocarcinomas which had ben fixed in buffered
 formalin and embedded in paraffin.

 The sections (10-30mm thick) were cut with
 standard microtome from every paraffin block and
 transferred into a 1,5 ml microtube.
DNA extraction :

 DNA extraction was performed using two
   different methods:

  a) a commercial kit (Dna extraction kit,
     BioDiagene) with a simple and rapid protocol

  b) a conventional method.
DNA extraction kit (BioDiagene)
• DNA was extracted from 10mm sections of
  paraffin embedded tissues.

• It is a rapid method consisting of xylene/ethanol
  dewaxing, followed by a kit base extraction
  without proteinase k digestion.
DNA extraction by conventional
method
• DNA was extracted from 10-30mm sections of
  paraffin embedded tissues.

• The conventional method consisted of
  xylene/ethanol dewaxing followed by overnight
  proteinase k digestion in lysis buffer. The sample
  was heated at 95°C for 5 minutes to inactivate the
  proteinase k.
Genomic DNA PCR

 We checked the quality of samples by PCR for the
 housekeeping gene b-globin (fragment of 268 bp).
 The PCR condition were as follows: denaturation at
 94°C for 5 minute, followed by 30 cycles of
 denaturation at 94°C for 1 minute, annealing at
 55°C for 1 minute and extension at 72°C for 1
 minute followed by 5 minutes final extension at
 72°C.
 The PCR amplification products were run on 2%
 agarose gel and stained with ethidium bromide and
 visualized under ultraviolet light.
              Results
Amplification of the b-globin gene sequence
was successful in 42 of 45 (93%) samples
using Dna extracted by commercial kit and
in 38 of 45 (84,4%) by conventional
methods (tab.1).
     Tab.1- Polymerase chain reaction (PCR)
 amplification of DNA (b-globin gene) extracted
from formalin fixed and paraffin embedded tissue
       Analysis by two different methods.


Methods          Total number of     PCR
                 samples amplified   success

Conventional         45              38 (84,4)
BioDiagene kit       45              43 (93%)
            Conclusion
Both methods used to obtain genomic DNA
from formalin fixed and paraffin-embedded
tissues produced DNA suitable for
amplification, but the rapid and non-
laborious commercial kit could facilitate
the molecular retrospective studies.
In fact, in only thirty minutes genomic DNA
is extracted and made ready to use for PCR
amplification.

				
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posted:3/1/2012
language:English
pages:11