scholarly journals DNA purification increases PCR-amplifiable DNA extracted from formalin-fixed, paraffin-embedded canine mast cell tumors for routine KIT mutation detection

2019 ◽  
Vol 31 (5) ◽  
pp. 756-760
Author(s):  
Vanessa S. Tamlin ◽  
Elizabeth C. Dobson ◽  
Lucy Woolford ◽  
Anne E. Peaston
Keyword(s):  
Mast Cell ◽  
Dna Extraction ◽  
Mutation Detection ◽  
Pcr Amplification ◽  
Low Grade ◽  
Kit Mutation ◽  
Mast Cell Tumors ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

DNA amplification by PCR detects KIT exon 11 internal tandem duplications in canine mast cell tumors (MCTs). Tissue-specific inhibitors often contaminate DNA extracted from formalin-fixed, paraffin-embedded (FFPE) canine MCTs, blocking PCR amplification and, consequently, preventing mutation detection. We used a commercial kit to extract DNA from FFPE canine MCTs. Two independent PCR assays, each with one primer set, were used to amplify target genes ( HPRT and KIT) directly after FFPE DNA extraction. PCR amplification failed with at least one primer set in 153 of 280 samples (54.6%, 95% CI: 48.8–60.5%). One or 2 DNA washing steps were required to remove PCR inhibitors in 130 of 280 (46.4%) and 23 of 280 (8.2%) of these cases, respectively. DNA concentration and quality (A260/A280 and A260/A230) either pre- or post-washing were not associated with ability of the samples to be amplified by PCR using both HPRT and KIT primer sets. Low-grade and subcutaneous MCTs were less likely to amplify directly after DNA extraction and without any washing steps compared to high-grade MCTs using KIT gene primers.

Reactivity of antibody YU-311 in formalin-fixed, paraffin-embedded specimens of normal organs, non-hematopoietic tumors, and mast cell tumors

1997 ◽  
Vol 47 (8) ◽  
pp. 512-517
Author(s):  
Takeaki Fukuda ◽  
Tomoko Kamishima ◽  
Toshio Kakihara ◽  
Toshiyuki Yamada ◽  
Kazuhito Matsumoto ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cell Tumors ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen Retrieval Principle: Heating Under the Influence of pH

2002 ◽  
Vol 50 (8) ◽  
pp. 1005-1011 ◽  
Author(s):  
Shan-Rong Shi ◽  
Richard J. Cote ◽  
Lin Wu ◽  
Cheng Liu ◽  
Ram Datar ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Control Sample ◽  
Pcr Amplification ◽  
Antigen Retrieval ◽  
Fixed Tissue ◽  
Ph Values ◽  
Formalin Fixed Paraffin ◽  
Paraffin Block ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6–9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6–12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.

scholarly journals Ancillary techniques on the evaluation of canine cutaneous mast cell tumors from Brazil

2016 ◽  
Vol 46 (10) ◽  
pp. 1804-1810 ◽  
Author(s):  
Mariana Martins Flores ◽  
Renata Dalcol Mazaro ◽  
Ingeborg Maria Langohr ◽  
Alma Roy ◽  
Keith Strother ◽  
...  
Keyword(s):  
Mast Cell ◽  
Pcr Amplification ◽  
The United States ◽  
Growth Fraction ◽  
Grading System ◽  
Low Grade ◽  
Ki 67 ◽  
Mast Cell Tumors ◽  
Cutaneous Mast Cell ◽  
Exon 11

ABSTRACT: The use of histologic classification by a 2-tier grading system only, immunohistochemistry (IHC) for KIT and Ki-67 and polymerase chain reaction (PCR) for internal tandem duplications (ITD) on exon 11 has improved the prognostication of canine cutaneous mast cell tumors (CCMTs) particularly in the United States. However, these techniques are not commonly used in most Brazilian laboratories. Likewise, no studies, to date, have investigated the occurrence of ITD in CCMTs from the country. Thus, this study tested the 2-tier grading system, the immunohistochemistry for KIT and Ki-67 and the PCR for exon 11 in a group of Brazilian CCMTs with the goal of investigating the applicability of these tests in a Brazilian laboratory. Of the 39 CCMTs, 69.2% (27/39) were identified as low-grade and 30.8% (12/39) as high-grade by a 2-tier grading system. All tumors had a KIT expression pattern II, and 30.6% (11/36) had a high growth fraction (Ki-67). PCR amplification was successful in four of the 11 tumors examined. Two of these (50%) were positive for ITD. This study highlights the importance of using auxiliary techniques in the CCMT evaluation, identifies limitations and confirms the applicability of these methods on a routine diagnostic basis in Brazil. Our results will help to improve the prognostication of CCMTs in Brazilian diagnostic laboratories, encouraging the use of supplementary methods.

scholarly journals Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tim A. D. Smith ◽  
Omneya A. AbdelKarem ◽  
Joely J. Irlam-Jones ◽  
Brian Lane ◽  
Helen Valentine ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Candidate Genes ◽  
Pcr Amplification ◽  
The Cancer Genome Atlas ◽  
Analysis Tool ◽  
Endogenous Control ◽  
Formalin Fixed Paraffin ◽  
Stability Ranking ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR.

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