Transcript An efficient and rapid method for DNA extraction from formalin

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
Conventional
BioDiagene kit
Total number of
samples amplified
45
45
PCR
success
38 (84,4)
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 nonlaborious 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.