An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues

Santos, Sara; Sá, Daniela; Bastos, Estela; Guedes-Pinto, Henrique; Gut, Ivo; Gärtner, Fátima; Chaves, Raquel

doi:10.1016/j.rvsc.2008.08.007

Cited by 44 publications

(39 citation statements)

References 38 publications

(45 reference statements)

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“…By comparing our DNA isolation strategy with those described in previous studies (Lin et al, 2009;Santos et al, 2009;Farrugia et al, 2010;Okello et al, 2010), our results indicate that the method of stocking frozen formalin fixation tissues for long periods of time is the major factor affecting the quality of genomic DNA. The pretreatment of the formalin fixation tissues by gradual dehydration can effectively reduce the crosslinking between histone proteins and DNA, even though formaldehyde cannot be completely removed (Fang et al, 2002).…”

Section: Discussionmentioning

confidence: 88%

“…The quality of the genomic DNA isolated from the frozen formalin fixation tissues was also evaluated in terms of its successful PCR amplification and sequencing results (Fang et al, 2002;Santos et al, 2009). The whole mtDNA control region (around 1589 bp) was amplified with 1 pair of primers (Zhao et al, 2009), and the whole mtDNA genome (around 16,569 bp) was amplified with two covering fragments, which were 8379 and 8604 bp long (Fendt et al, 2009) (Figure S1b and S1c).…”

Section: Resultsmentioning

confidence: 99%

“…Formalin-fixed paraffin-embedded (FFPE) tissue contains a rich source of genetic material that can be used for medical research (Lipson et al, 2012;Chen et al, 2013) and biological disciplines (Santos et al, 2009) in downstream DNA-based experiments, such as copy number analysis (Gilbert et al, 2007;Jacobs et al, 2007), real-time quantitative polymerase chain reaction (RT-PCR) (Gjerdrum et al, 2004), single nucleotide polymorphism (SNP) bead arrays (Oosting et al, 2007), microarray comparative genomic hybridization (Vékony et al, 2007), and genome-wide sequencing (Aviel-Ronen et al, 2006;Schumer et al, 2013). However, formalin may cause the fragmentation of DNA (Douglas and Rogers, 1998), which reduces the quality and quantity of DNA (Poljak et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…Fortunately, the formation of these crosslinking is reversible in aqueous solution (Dubeau et al, 1986;Fang et al, 2002). Thus, the possibility of obtaining high-quality genomic DNA from FFPE tissue was presented by performing a pre-extraction procedure to remove paraffin from fixed tissues (Santos et al, 2009). Previous study has confirmed the superiority of genomic DNA after pretreatment of the FFPE tissues (Fang et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Improved protocol for extracting genomic DNA from frozen formalin-fixed tissue resulting in high-quality whole mtDNA

Wang

Zhang

et al. 2016

Genet. Mol. Res.

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ABSTRACT. Formalin fixation and paraffin embedding is widely used for convenient and long-term storage of tumor tissue and precious sources to perform genetic studies. However, DNA fragmentation is one of the major flaws of genomic DNA isolation from formalin fixation tissues, which limits its further usage. Here, we present an improved method for isolating high-quality genomic DNA from formalin fixation tissue. We obtained high-quality genomic DNA of more than 20 kb from samples frozen for more than 2 years. Furthermore, to verify DNA quality, the whole mitochondrial DNA (mtDNA) genomes from the normal and tumor tissue of the same patient were successfully amplified with two overlapping PCR fragments comprising more than 8379 bp in length for each fragment. In addition, the whole genomes were sequenced with a 48-well based primer panel in order to avoid potential sequencing errors from artificial recombination, which was further confirmed with an mtDNA phylogenetic strategy. Our improved DNA extraction method from formalin fixation tissue and sequencing strategy for entire mtDNA genomes will generate unambiguous sequence analysis results for clinical samples.

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Section: Discussionmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Improved protocol for extracting genomic DNA from frozen formalin-fixed tissue resulting in high-quality whole mtDNA

Wang

Zhang

et al. 2016

Genet. Mol. Res.

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“…Sin embargo, los largos periodos de tiempo en que permanecen las estructuras anatómicas promueven que el Formaldehido se transforme gradualmente en Ácido Fórmico, el cual produce entrecruzamiento (cross-linkange) entre ácidos nucléicos y proteínas; hidroliza puentes fosfodiester del DNA y modifica covalentemente el RNA por adición de grupos metilos en las bases nitrogenadas, generando problemas en la obtención de material genético óptimo para realizar posteriores estudios moleculares (2,3,4). No obstante, estudios han demostrado que el tratamiento con proteinasas mejora la recuperación del DNA, ya que permite liberar los ácidos nucléicos atrapados en los complejos proteicos (5) y han tratado de mejorar la digestión del tejido mediante el aumento de la concentración de proteinasa K, prolongación del período de digestión, y/o mediante el aumento de la temperatura de incubación (3,6,7,8).…”

Section: Introductionunclassified

Evaluación de un método de extracción y purificación de DNA a partir de tejido muscular fijado en formaldehido de cadáveres no identificados

et al. 2014

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ResumenLa obtención de DNA humano en buena cantidad y alta pureza a partir de tejido muscular fijado en Formaldehido no tamponado es de suma importancia en la amplificación por la Reacción en Cadena de la Polimerasa (PCR) para su aplicación en estudios de identificación y filiación genética. En este estudio se evaluó la eficiencia del Kit QIAampR DNA FFPE TISSUE y una modificación del mismo basado en lavados con PBS y el tiempo de digestión con proteinasa K, frente a la cantidad y calidad de este ácido nucléico. Las diferencias fueron significativas entre los tiempos de acción con la proteinasa K (PK) en relación a la cantidad y en la pureza producto de los lavados del tejido muscular previa extracción. Estos resultados proporcionan una pauta para el diseño de experimentos de acuerdo con el efecto de la fijación, optimizando recursos humanos e insumos.Palabras clave: Formaldehido, tejido muscular, proteinasa K, lavado celular. EVALUATION OF A METHOD OF EXTRACTING AND PURIFYING DNA FROM MUSCLE TISSUE FIXED IN FORMALDEHYDE UNIDENTIFIED BODIES AbstractObtaining human DNA in large quantity and high purity from muscle tissue fixed in formaldehyde unbuffered is of utmost importance in amplification by Chain Reaction (PCR) for application in identification studies and genetic affiliation. In this study, the efficiency of DNA FFPE TISSUE QIAampR Kit and a modification thereof based washes with PBS and the time of digestion with proteinase K, compared to the amount and quality of the nucleic acid were evaluated. Differences were significant between the exposure times with proteinase K (PK) in relation to the quantity and purity of the product prior washings muscle tissue extraction. These results provide a guideline for the design of experiments according to the effect of fixing, optimizing human resources and inputs.

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Reducing DNA damage by formaldehyde in liquid‐based cytology preservation solutions to enable the molecular testing of lung cancer specimens

Matsuo

Yoshida

Yamashita

et al. 2018

Cancer Cytopathology

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Background Liquid‐based cytology (LBC) is a useful cytopathological method, and LBC lung adenocarcinoma specimens may be used for genetic analysis in the near future. In the current study, the authors determined whether LBC specimens can be used for epidermal growth factor receptor (EGFR) mutation analysis in human lung adenocarcinoma cell lines. Methods Genomic DNA was extracted from 3 lung adenocarcinoma cell lines that were fixed in LBC preservation solution using 2 protocols (one for cultured cells and one for tissues) of a DNA extraction kit. Different fixation times were tested for each protocol: 30 minutes, 1 hour, and 1 to 9 days. As controls, cells also were fixed in 10% formalin or 95% ethanol. The authors investigated the effect of fixation time on DNA fragmentation, polymerase chain reaction (PCR) amplification, and EGFR mutation detection. Results The DNA yield of LBC specimens tended to decrease depending on fixation time. When using the DNA extraction protocol for tissues, PCR amplification was successful after 9 days of fixation, although extracted genomic DNA that was fixed for >1 hour demonstrated fragmentation. Mutation analyses using the Cycleave PCR method were successful after 7 days of fixation. The DNA extraction protocol for tissues was appropriate for lung adenocarcinoma cell lines that were stored for >1 day in a preservative solution. The results of the current study demonstrated that EGFR mutations can be detected on day 7 using lung adenocarcinoma cell lines fixed in CytoRich Red preservative. Conclusions When LBC specimens are used for targeted molecular genetic testing, the appropriate preservative solution and extraction protocol first should be determined.

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An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues

Cited by 44 publications

References 38 publications

Improved protocol for extracting genomic DNA from frozen formalin-fixed tissue resulting in high-quality whole mtDNA

Improved protocol for extracting genomic DNA from frozen formalin-fixed tissue resulting in high-quality whole mtDNA

Evaluación de un método de extracción y purificación de DNA a partir de tejido muscular fijado en formaldehido de cadáveres no identificados

Reducing DNA damage by formaldehyde in liquid‐based cytology preservation solutions to enable the molecular testing of lung cancer specimens

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