Extraction and Amplification of DNA From Formalin-Fixed, Paraffin-Embedded Tissues

Wu, Lin; Patten, Nancy; Yamashiro, Carl T.; Chui, Buena

doi:10.1097/00129039-200209000-00015

Cited by 62 publications

(56 citation statements)

References 11 publications

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“…Indeed, while the routine fixative formalin preserves the tissue morphology, formalin fixation of tissues can cause the formation of protein-DNA cross-links, limiting the analysis of nucleic acids (Wu et al, 2002). The amplification products can thus be reduced in quantity and size compared to fresh or frozen tissues (Greer et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden infant death victims

et al. 2004

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Previous studies have provided histological evidence of an association between primary Pneumocystis infection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clustered Pneumocystis organisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derived Pneumocystis. Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of the Pneumocystis species using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding to Pneumocystis carinii were found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims. Pneumocystis jirovecii was successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that the Pneumocystis species found in the lungs of Chilean SID victims was P. jirovecii.

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Section: Discussionmentioning

confidence: 99%

Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden infant death victims

et al. 2004

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“…The half-life of formaldehyde crosslinks is inversely correlated with temperature (48 ). High-temperature heating methods, usually at Ͼ90°C, have been shown to be effective not only for the yield of DNA (50 ), but also for the yield of amplifiable templates from FFPE tissues (49,51 ).…”

Section: Removal Of Crosslinks By Heat Treatmentmentioning

confidence: 99%

Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

2015

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BACKGROUND: Precision medicine is dependent on identifying actionable mutations in tumors. Accurate detection of mutations is often problematic in formalinfixed paraffin-embedded (FFPE) tissues. DNA extracted from formalin-fixed tissues is fragmented and also contains DNA lesions that are the sources of sequence artifacts. Sequence artifacts can be difficult to distinguish from true mutations, especially in the context of tumor heterogeneity, and are an increasing interpretive problem in this era of massively parallel sequencing. Understanding of the sources of sequence artifacts in FFPE tissues and implementation of preventative strategies are critical to improve the accurate detection of actionable mutations.CONTENT: This mini-review focuses on DNA template lesions in FFPE tissues as the source of sequence artifacts in molecular analysis. In particular, fragmentation, base modification (including uracil and thymine deriving from cytosine deamination), and abasic sites are discussed as indirect or direct sources of sequence artifacts. We discuss strategies that can be implemented to minimize sequence artifacts and to distinguish true mutations from sequence artifacts. These strategies are applicable for the detection of actionable mutations in both single amplicon and massively parallel amplicon sequencing approaches.

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“…For each sample, a tumorenriched region was identified by a pathologist and macrodissected from 5-m-thick tissue sections. The macrodissected tissues were incubated at 98°C for 15 min before undergoing proteinase K digestion for 3 days (16 ). We then extracted genomic DNA using the DNeasy Tissue and Blood kit (Qiagen) according to the manufacturer's protocol.…”

Section: Processing Of Ffpe Dnamentioning

confidence: 99%

Reducing Sequence Artifacts in Amplicon-Based Massively Parallel Sequencing of Formalin-Fixed Paraffin-Embedded DNA by Enzymatic Depletion of Uracil-Containing Templates

Wong

et al. 2013

Clinical Chemistry

110

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BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for detecting mutational biomarkers in patients with cancer. A previous intractable challenge with FFPE DNA in genetic testing has been the high number of artifactual single-nucleotide changes (SNCs), particularly for the detection of lowlevel mutations. Pretreatment of FFPE DNA with uracil-DNA glycosylase (UDG) can markedly reduce these C:GϾT:A SNCs with a small panel of amplicons. This procedure has implications for massively parallel sequencing approaches to mutation detection from DNA. We investigated whether sequence artifacts were problematic in amplicon-based massively parallel sequencing and what effect UDG pretreatment had on reducing these artifacts.

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Extraction and Amplification of DNA From Formalin-Fixed, Paraffin-Embedded Tissues

Cited by 62 publications

References 11 publications

Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden infant death victims

Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden infant death victims

Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

Reducing Sequence Artifacts in Amplicon-Based Massively Parallel Sequencing of Formalin-Fixed Paraffin-Embedded DNA by Enzymatic Depletion of Uracil-Containing Templates

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