Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue

Potluri, Keerti; Mahas, Ahmed; Kent, M.; Naik, Sameep; Markey, Michael P.

doi:10.1016/j.ab.2015.06.029

Cited by 20 publications

(9 citation statements)

References 24 publications

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“…Because the majority of FFPE DNA/RNA extraction kits were launched in the last few years, they generally lack documented performance in peer-reviewed literature. However, recent headto-head comparison studies suggest that these kits might differ significantly in terms of DNA yield, purity, and quality (12,18). Therefore, it seems that the transition to one of the available FFPE DNA/RNA commercial kits will not be so straightforward and will require extensive comparisons with the established lab protocol in advance.…”

Section: Discussionmentioning

confidence: 99%

Commercially available kits for manual and automatic extraction of nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissues

Kocjan¹,

Hošnjak²,

Poljak³

2015

Acta Dermatovenerologica Alpina Pannonica Et Adriatica

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Introduction: Formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable source for diagnostic purposes when fresh clinical material is unavailable, and also for molecular and epidemiological studies. The recovery of nucleic acids from FFPE tissues is particularly challenging, and several in-house methods have been developed for this purpose over the last three decades. Recently, several commercial kits specifically developed for DNA and/or RNA extraction from FFPE tissues have been introduced to the market, but their inventory is not available in peer-reviewed literature. Methods: This article provides the first comprehensive inventory of commercial FFPE DNA/RNA extraction kits currently available on the market and describes their basic characteristics and features. Results: A total of 69 commercial kits from 43 companies were identified. Thirty-five kits were developed specifically for DNA extraction, 22 for RNA extraction, and 12 for both DNA and RNA extraction. Only two commercial kits allow full automation of the entire nucleic acid extraction procedure. The tissue deparaffinization step is omitted in many protocols by melting paraffin directly in a tissue lysis buffer. Purification of the released nucleic acids is mainly based on silica or resin adsorption technology. A formalin reverse cross-linking step to increase the quality of extracted DNA and RNA is an intrinsic part of over half of the kits identified. Conclusions: It is hope that this comprehensive list of available commercial kits for extracting nucleic acids from FFPE will encourage researchers to strongly consider using them in diagnostic and research settings instead of old-fashioned, crude, and probably less effective in-house methods.

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Section: Discussionmentioning

confidence: 99%

Commercially available kits for manual and automatic extraction of nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissues

Kocjan¹,

Hošnjak²,

Poljak³

2015

Acta Dermatovenerologica Alpina Pannonica Et Adriatica

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“…However, such sensitivity might be affected considering the use of clinical samples that are usually complicated with the presence of PCR interfering components in the samples. Nevertheless, most interfering components could be eliminated by DNA purification using FFPE tissue kits as we employed in this study and as previously described [ 46 , 47 ]. Moreover, the MAS-PCR assay that we developed has a higher sensitivity than that reported for direct sequencing and HRM (i.e., 5% to 20%), and is equivalent to that reported for pyrosequencing and commercial molecular kits (i.e., 1% to 5%) [ 14 , 18 , 38 ].…”

Section: Discussionmentioning

confidence: 99%

Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients

2016

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BackgroundPatients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations.MethodsWe developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.ResultsOverall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12.ConclusionThe MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

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“…The amplified PCR product was excised and eluted from the gel using Wizard ® SV Gel and PCR Clean-Up System (Promega, USA). Of the 20 paraffin blocks of insulinomas retrieved from the Department of Pathology, DNA from 3 samples could not be amplified successfully in PCR owing to either low DNA yield or sheared DNA quality, even after repeating DNA isolation [21,22]. We, therefore, were able to sequence YY1 exon 5 in only 17 out of 20 samples.…”

Section: Mutation Analysismentioning

confidence: 99%

T372R Mutation Status in Yin Yang 1 Gene in Insulinoma Patients

et al. 2017

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Insulinomas are rare pancreatic neuroendocrine tumors. The genetic causes underlying insulinoma are still being investigated. Recently, 3 independent studies reported a recurrent somatic mutation in YY1 gene (C>G; Thr372Arg) among insulinoma patients belonging to Chinese and Western Caucasian populations, which was found to increase insulin secretion by β-cells. However, the status of this key gene variation remains unknown in patients of other ethnicities. We, therefore, screened Indian sporadic insulinoma patients for YY1 T372R mutation in the present study. Seventeen patients diagnosed with insulinoma were recruited retrospectively and their records of family history and clinical parameters were collected. Formalin-fixed paraffin-embedded tumor tissues were used to extract genomic DNA, which was subjected to PCR amplification of YY1 exon 5, followed by Sanger sequencing. Nucleotide sequences thus obtained were aligned against the documented sequence of YY1 exon 5. We found absence of C to G mutation at YY1 codon 372 in all 17 (100%) insulinoma tissues analyzed. On comparison with the mutation frequency observed in the Chinese patients, our results point to genetic heterogeneity in the pathogenesis of insulinoma. This is the first report on the status of YY1 T372R in insulinoma cases of Indian origin. This also warrants analysis of other documented as well as novel mutations in genes in insulinoma tumorigenesis.

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Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue

Cited by 20 publications

References 24 publications

Commercially available kits for manual and automatic extraction of nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissues

Commercially available kits for manual and automatic extraction of nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissues

Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients

T372R Mutation Status in Yin Yang 1 Gene in Insulinoma Patients

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