Improved protocol for extraction of genomic DNA from formalin-fixed paraffin-embedded tissue samples without the use of xylene

AimsTo evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched PCR followed by reverse-hybridisation of biotinylated amplification products to oligonucleotide probes immobilised as an array of parallel lines on nitrocellulose test strips.MethodsThe strip assay was used to analyse genomic DNA isolated from 120 formalin-fixed paraffin-embedded (FFPE) ovarian tissue samples. The samples were analysed in parallel using a biochip-based protocol (biochip assay) covering the same mutation spectrum, and results were compared with respect to sensitivity, specificity and operational input.ResultsThe strip assay identified 19 (16%) of 120 FFPE samples to carry a KRAS mutation; results were in agreement with those obtained by biochip hybridisation. Both assays had an analytical sensitivity of 1% when performed on FFPE-extracted DNA with approximately the same operational input needed for post-PCR processing. In contrast to the biochip assay, strip assay hybridisation may be automated to a large extent.ConclusionsThe strip assay is an accurate and sensitive tool for the low to medium throughput detection of KRAS mutation in genomic DNA isolated from FFPE tissue.

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A High-Temperature, Pre-Incubation Step Before Proteinase K Treatment Notably Improves Recovery of Genomic DNA in Formalin-Fixed, Paraffin-Embedded Tissue Samples

Clinical Laboratory ◽

10.7754/clin.lab.2020.200432 ◽

2020 ◽

Vol 66 (12/2020) ◽

Author(s):

Arizumi Kikuchi ◽

Azumi Naruse ◽

Takahiro Sawamura ◽

Kenichi Nonaka

Keyword(s):

High Temperature ◽

Genomic Dna ◽

Proteinase K ◽

Tissue Samples ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Proteinase K Treatment ◽

Formalin Fixed

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Evaluation of three methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA by means of the PCR technique

Pesquisa Odontológica Brasileira ◽

10.1590/s1517-74912001000400008 ◽

2001 ◽

Vol 15 (4) ◽

pp. 314-318 ◽

Cited By ~ 9

Author(s):

Ricardo Alves MESQUITA ◽

Evelyn K. ANZAI ◽

Rogério Nogueira OLIVEIRA ◽

Fábio Daumas NUNES

Keyword(s):

Genomic Dna ◽

Retrospective Studies ◽

Enzymatic Digestion ◽

Tissue Samples ◽

Normal Oral Mucosa ◽

Formalin Fixed Paraffin ◽

Histopathologic Diagnosis ◽

Formalin Fixed Paraffin Embedded ◽

Digestion Methods ◽

Formalin Fixed

There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

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The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use

Cell and Tissue Banking ◽

10.1007/s10561-021-09949-1 ◽

2021 ◽

Author(s):

Robin Verjans ◽

Annette H. Bruggink ◽

Robby Kibbelaar ◽

Jos Bart ◽

Aletta Debernardi ◽

...

Keyword(s):

The Netherlands ◽

Tissue Sample ◽

Ffpe Tissue ◽

Tissue Samples ◽

Internet Portal ◽

Formalin Fixed Paraffin ◽

Practical Support ◽

Formalin Fixed Paraffin Embedded ◽

The Rich ◽

Formalin Fixed

AbstractBiobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation—a nationwide network and registry of histo- and cytopathology in the Netherlands—was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA’s nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use. We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA’s nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (− 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA’s national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands.

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Detection of clonality in follicular lymphoma using formalin-fixed, paraffin-embedded tissue samples and BIOMED-2 immunoglobulin primers

Journal of Clinical Pathology ◽

10.1136/jcp.2010.081109 ◽

2010 ◽

Vol 64 (1) ◽

pp. 37-41 ◽

Cited By ~ 28

Author(s):

E. Berget ◽

L. Helgeland ◽

A. Molven ◽

O. K. Vintermyr

Keyword(s):

Follicular Lymphoma ◽

Tissue Samples ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Microsatellite instability evaluation: which test to use for endometrial cancer?

Journal of Clinical Pathology ◽

10.1136/jclinpath-2021-207723 ◽

2021 ◽

pp. jclinpath-2021-207723

Author(s):

Paola Rafaniello-Raviele ◽

Ilaria Betella ◽

Alessandra Rappa ◽

Davide Vacirca ◽

Gianluca Tolva ◽

...

Keyword(s):

Endometrial Cancer ◽

Microsatellite Instability ◽

Automated System ◽

Tissue Samples ◽

Valuable Alternative ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed ◽

Mmr Proteins

AimsAnalysis of microsatellite instability (MSI) is strongly recommended in endometrial cancer (EC) and colorectal cancer to screen for Lynch syndrome, to predict prognosis and to determine optimal treatment and follow-up. In a large monoinstitutional series of ECs, we evaluated the reliability and accuracy of Idylla assay, a rapid, fully automated system to detect MSI, and we compared its performance with two routine reference methods.MethodsWe evaluated MSI status in 174 formalin-fixed, paraffin-embedded EC tissue samples using immunohistochemistry (IHC) for mismatch repair (MMR) proteins and Idylla assay. Samples with discordant or equivocal results were analysed with a third technique, the Promega MSI kit.ResultsIdylla MSI assay and IHC were highly concordant (overall agreement: 154/170=90.59%, 95% CI 85.26% to 94.12%). However, in four samples, MMR-IHC staining was equivocal; moreover, 16 cases showed discordant results, that is, MMR deficient using IHC and microsatellite stable using Idylla. These 20 samples were reanalysed using the MSI-Promega kit, which showed the same results of Idylla assay in 18/20 cases (overall agreement: 90%, 95% CI 69.90% to 97.21%).ConclusionsOur results suggest that IHC is an efficient method to determine MMR status in ECs. However, the Idylla MSI assay is a rapid and reliable tool to define MSI status, and it could represent a valuable alternative to conventional MSI-PCR methods.

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