Rapid Salt-Extraction of Genomic DNA from Formalin-Fixed Toad and Frog Tissues for PCR-Based Analyses
The technical procedures for extraction of DNA from formalin-fixed tissues include many steps such as chemical treatment, enzymatic digestion, phenol-chloroform purification and alcohol precipitation. Formalin-fixed specimens used in molecular cell and DNA studies have shown shortcomings with respect to the efficacy of DNA isolation and subsequent PCR (Polymerase Chain Reaction) amplification. This study was designed to simplify and maximize recovery of PCR-amplifiable DNA from formalin-fixed toad and frog specimens and also to minimize co-extraction of substances that inhibit PCR amplification. This is achieved by a combination of DNA extraction from formalin-fixed muscle tissues using a salt-out buffer consisting of EDTA and proteinase K and NaCl. All steps are performed at room temperature (20-25°C), thereby reducing further degradation of the already damaged fragile specimen DNA and providing an optimal trade-off between DNA release and degradation. The salt-extraction method of genomic DNA presented here allows DNA isolation from formalin-fixed tissues with a minimum of working steps and equipment and rapidly yields much DNA.
Received: May 15, 2011;
Accepted: June 29, 2011;
Published: August 08, 2011
Molecular phylogenetics, behavioral ecology and population biology has increased
dramatically during the last decades. For the application of these techniques,
it is essential to obtain tissue samples allowing proper extraction of nucleic
acids. Genomic DNA or RNA usually obtained from fresh or frozen tissues. Although
the extraction of high-quality nucleic acid may be problematic from formalin-fixed
tissues because of cross-linking between DNA and proteins or impurities, there
are many studies on PCR-based analysis using formalin-fixed tissues have been
published by Jackson et al. (1998), Harty
et al. (2000), Lewis et al. (2001), Specht
et al. (2001), Drabkova et al. (2002),
Shi et al. (2002, 2004),
Cao et al. (2003), Bibikova
et al. (2004), Bahador et al. (2004),
Rivero et al. (2006), Negishi
et al. (2009) and April et al. (2009).
The Polymerase Chain Reaction (PCR) is an in vitro amplification technique
that depends on adequate storages of samples and good protocols for DNA extraction.
Methods for DNA extraction from fresh tissue and cytological preparation have
been described and adapted for use in some archival specimens (Shibata
et al., 1988; Coates et al., 1991; Akao
et al., 1991; Foss et al., 1994; Frank
et al., 1996; Mao et al., 1996; Adams
et al., 1996; Diaz-Cano and Brady, 1997).
The most common archival specimens are formalin-fixed and/or Paraffin-Embedded
Tissues (PETs). DNA can be extracted from PETs but archival tissues may be unsuitable
for many molecular techniques which require high molecular weight genomic DNA,
as slow degradation of DNA occurs with time. However, short segments of genomic
DNA are useful as a substrate for PCR amplification (Foss
et al., 1994; Mies, 1994; Diaz-Cano
and Brady, 1997; Akalu and Reichardt, 1999; Sato
et al., 2001; Drabkova et al., 2002;
Cao et al., 2003) and many researchers have also
shown that PCR can be performed successfully on nucleic acids (DNA or RNA) that
are partially degraded over time.
Formalin is the most acceptable fluid for soft tissue preservation and is by
far one of the most widely fixatives used in specimen collections, particularly
for toads and frogs. Formalin has been used as a fixative in archival specimens
for more than a hundred years. During the research practice of last century,
a large number of formalin-fixed tissue banks have been established. These tissue
banks form invaluable resources of samples for various translational studies
of molecular genetics and evolution and other interesting topics. The accessibility
of macromolecules in fixed tissue specimens is a critical issue, as exemplified
by the growth of PCR-based analyses. Although several DNA extraction methods
for formalin-fixed and/or paraffin-embedded tissues were previously proposed
by Rogers et al. (1990), Stein
and Raoult (1992), Forsthoefel et al. (1992),
Freeman et al. (1997), Merkelbach
et al. (1997), Lum and Marchand (1998), Mulot
et al. (2005), Huang et al. (2005), Bremmer
et al. (2005), Cao et al. (2003) and
Rivero et al. (2006), few studies have been conducted
to compare these existing methods in order to identify a better method for DNA
isolation. The present study was designed to rapidly isolate genomic DNA with
salt-extraction method from formalin-fixed tissues of toads and frogs. We also
evaluated the quality of genomic DNA extracted from toads and frogs fixed for
five to ten years with PCR amplification.
MATERIALS AND METHODS
Tissues and DNA extraction: The study was conducted from October, 2009 to December, 2010. Formalin-fixed toad and frog muscle tissues were obtained from the Experimental Center of Fuyang Normal College from 2001 to 2010. All samples were routinely fixed in 10% neutral buffered formalin (average period of fixation was 24 h at room temperature, 20-25°C). All fixed tissues were processed routinely as required by the Experimental Center. All protocols were approved by the Institutional Review Board and the Institutional Animal Care and Use Committee of Fuyang Normal College.
The muscles of one back leg and/or part body of a toad or frog were used for
DNA extraction. DNA was extracted using a modified salt-extraction method (Aljanabi
and Martinez, 1997; Sambrook, 2001; Rivero
et al., 2006). The formalin-fixed muscle tissue was homogenized in
450 mL of sterile salt homogenizing buffer (0.4 M NaCl 10 mM Tris-HCl pH 8.0
and 2 mM EDTA pH 8.0) for 10-15 s. Then, 40 mL of 20% SDS (2% final concentration)
and 8 mL of 10 mg mL-1 protenase K (200 mg mL-1 final
concentration) were added and mixed well. The samples were incubated at 55-56°C
overnight, after which 300 mL of 6 M NaCl solution (NaCl saturated H2O)
was added to each sample. Completed genomic DNA extraction was performed by
the salt-extraction method, according to Aljanabi and Martinez
(1997). Genomic DNA purity was assessed with a spectrophotometer and calculated
by the ratio of DNA optical density (A260) and protein optical density (A280).
Genomic DNA yield was calculated from DNA optical density (OD 260) for clean
DNA samples. The purity of genomic DNA, determined from the A260/A280 ratio
was averaged >1.71 for all samples. There was no RNA contamination in all
samples during preparation.
|| Primers of 12 S rRNA used in this study
Following extraction, 4 to 5 mL samples were run on agarose electrophoresis gels (1%) containing ethidium bromide, with a 15000 bp ladder (marker D15000+2000) and were photographed under UV light to estimate the size range of genomic DNA fragments. Later, 1-2 μL DNA was used for PCR amplifications. The amount of tissue required for this method is minimal and the average number of PCR amplifications that can be performed using DNA extracted from 50 mg tissue was >1000.
PCR analysis: Each DNA extract was used as a template for PCR amplification, using a primer pair of 12 S rRNA genes as listed in Table 1. PCR tests were carried out based on groups of DNA samples extracted from eight formalin-fixed muscle tissues and a total of 16 PCR test results were evaluated by gel electrophoresis. PCR was performed by standard protocols. Briefly, the DNA sample diluted in 1-2 μL of distilled water containing 100 ng as template was added to the PCR reaction Mixture. PCR amplifications were carried out in a total volume of 25 μL. The PCR reaction mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.25 mM of dNTP each, 0.01 mg Bovine Serum Albumin (BSA), 50 ng of each primer, 0.05 units of Taq polymerase and about 50 ng of genomic DNA.
The PCR amplification program was designed with an initial denaturing step at 94°C for 5 min, followed by 35 cycles at 94°C for 30 sec, 52°C for 40 sec and 72°C for 30 sec, with a final hold at 4°C for 10 min to complete the program. The PCR amplification products were then discerned by electrophoresis on 2% agarose gels for about 30 min at 120 V and stained with ethidium bromide for visualization under UV light.
RESULTS AND DISCUSSION
The total DNA extracted from formalin-fixed toad and frog tissues were 14 out
of 16 specimens examined, as shown in Fig. 1a and b.
The toad DNA was successfully extracted from 8/8 samples (Fig.
1b) with about two folds of the amount of frog muscle tissues used in Fig.
1a. The failures were the two older samples, collected 10 years ago.
All the samples of genomic DNA extracted from formalin-fixed toad and frog tissues had been PCR amplified (Fig. 2). The PCR products amplified of toad and frog 12 S rRNA genes were a fragment of 350 bp. Toad 12S rRNA gene were sequenced successfully for 15 out of 16 PCR products (Fig. 2b). The PCR products were shown as specific. Frog samples 12S rRNA gene were sequenced successfully for all PCR products but some unspecific DNA bands appeared (Fig. 2a). The results revealed that the genomic DNA extracts from formalin-fixed tissues of toad and frog were about 10000 bp (Fig. 1) and none of PCR negative controls or extraction blanks exhibited signs of contamination with RNA or fungi DNA (Fig. 2).
As demonstrated in Fig. 1 and 2, the samples
exhibited no significant external change/damage post extraction and the PCR
products were satisfying (Fig. 2). All genomic DNA extracts
produced a clear, sharp and reproducible PCR amplification product pattern.
We had the same results after we repeated the PCR experiment over a period.
As regards to these results, the modified salt-extraction method was validated
for DNA extraction from formalin-fixed tissues.
|Fig. 1 (a-b):
||(a) Frog and (b) Toad DNA extracts from formalin-fixed muscle
tissues, discerned by electrophoresis on 1% agarose gels with a 15000-bp
ladder (D15000+2000, noted as M). Marker B shows blank lanes working as
|Fig. 2 (a-b):
||PCR products of 12S rRNA gene sequences from formalin-fixed
tissues of (a) Frog and (b) Toad, amplified with the primers 12S1091/12S1092,
were discerned by electrophoresis on 2% agarose gels with a 2000-bp ladder
(DL2000, noted as M). Marker B shows blank lanes working as negative control
These results were in agreement with Drabkova et al.
(2002) and Cao et al. (2003). Although the
methods used in their reports were not salt-based types. The present study also
examined the efficacy of salt-extraction method derived from Aljanabi
and Martinez (1997) and Rivero et al. (2006).
In the present study, sufficient DNA of samples was retrieved to enable us to
provide enough DNA for PCR-based analyses.
Pervious methods for sample preparation of DNA from formalin-fixed and/or paraffin-embedded
tissues are time consuming. These methods involved many steps and require several
centrifugations and washes and multiple tube transfers which increase opportunities
for the introduction of contaminant (Shibata et al.,
1988; Rogers et al., 1990; Stein
and Raoult, 1992; Forsthoefel et al., 1992;
Freeman et al., 1997; Huang
et al., 2005).
Most of the recent studies used the DNA extraction with modified phenolchloroform
protocol, boiling method and commercial DNA Extraction Kit (Shi
et al., 2002, 2004; Drabkova
et al., 2002; Cao et al., 2003). However,
there are few reports of DNA extraction methods similar to our protocol. Rivero
et al. (2006) studied a simple method of DNA extraction from formalin-fixed
and paraffin-embedded tissues using a salt solution to precipitate protein and
isopropanol to precipitate DNA. They focused on samples from Paraffin-Embedded
Tissues (PETs). Their samples were tissues from small biopsies of three oral
Inflammatory Fibrous Hyperplasia (IFH) and three oral Squamous Cell Carcinomas
(SCC), first fixed in 10% buffered formalin and then embedded in paraffin. They
compared the salting-out DNA extraction method with a phenolchloroform
extraction method and a commercial DNA isolation kit. According to their results,
the extraction method using proper concentrations of ammonium acetate proved
to be simple and suitable for obtaining high quality DNA.
Usually, blood and leaf samples have been the specimens of choice for genomic
DNA in molecular genetics and/or molecular biology studies (Bahador
et al., 2004; Eshraghi et al., 2006;
Bailes et al., 2007; Khairalla
et al., 2007; Dehestani and Kazemi Tabar, 2007;
Sahasrabudhe and Deodhar, 2010; Shankar
et al., 2011; Chaudhary et al., 2011).
Various methods are currently available to extract DNA from blood lymphocytes
and other animal tissues with phenol-chloroform (Bailes
et al., 2007; Khairalla et al., 2007;
Chamani-Tabriz et al., 2007). However, collecting
these samples is invasive and expensive and none of DNA extraction methods are
ideal or universal. In comparison with phenol-chloroform based methods, salt-extraction
or salt-out method is relatively simple, feasible, rapid and more acceptable
by museum and field research participants (Drabkova et
al., 2002; Cao et al., 2003; Aljanabi
and Martinez, 1997; Rivero et al., 2006).
The use of molecular techniques on archival materials has been limited due to
the difficulty in obtaining consistent results. It is accepted that genomic
DNA extracted from formalin-fixed and PETs archived specimens is not well preserved
or is degraded but some molecular techniques require high molecular weight DNA
(Shi et al., 2002, 2004;
Drabkova et al., 2002; Cao
et al., 2003; Bahador et al., 2004;
Rivero et al., 2006; April
et al., 2009). The reasons why formalin-fixed and PETs undergo degradation
include insufficient neutralization of the formalin, causing acid depurination
of DNA and preventing amplification (Shibata et al.,
1988; Drabkova et al., 2002; Cao
et al., 2003; Bahador et al., 2004).
Present results showed that despite degradation, it is possible to use the genomic
DNA from formalin-fixed for the past five to ten years in PCR amplification
of short specific gene sequences. In the present study, amplification of a 350
bp fragment of 12 S rRNA genes was successful in all the 16 samples extracted
by the salt-extraction method. Another important problem is the toxicity of
phenol. Procedures using salt have been used to extract DNA from blood and other
samples and were proved to be less laborious and non-toxic than the phenol-chloroform
techniques. This makes the method an attractive optional method of genomic DNA
Present results proved that the modified simple salt-extraction method was considered proper and satisfying as one of the rapid methods for DNA extraction from formalin-fixed archival specimens or tissues.
We are grateful to the anonymous reviewers for their constructive comments and suggestions. This work was supported by Chinese grants from Anhui Educational Research Funds to LWY (2005QL11, 2006jql222, 2006KJ224B).
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