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DNA purification increases PCR-amplifiable DNA extracted from formalin-fixed, paraffin-embedded canine mast cell tumors for routine KIT mutation detection.
- Source :
-
Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc [J Vet Diagn Invest] 2019 Sep; Vol. 31 (5), pp. 756-760. Date of Electronic Publication: 2019 Aug 04. - Publication Year :
- 2019
-
Abstract
- 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 (A <subscript>260</subscript> /A <subscript>280</subscript> and A <subscript>260</subscript> /A <subscript>230</subscript> ) 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.
- Subjects :
- Animals
Dogs
Formaldehyde
Mast Cells cytology
Mastocytoma diagnosis
Mutation
Nucleic Acid Amplification Techniques methods
Paraffin Embedding veterinary
Polymerase Chain Reaction methods
Proto-Oncogene Proteins c-kit analysis
Skin Neoplasms diagnosis
Skin Neoplasms veterinary
Circulating Tumor DNA analysis
Dog Diseases diagnosis
Mastocytoma veterinary
Nucleic Acid Amplification Techniques veterinary
Polymerase Chain Reaction veterinary
Subjects
Details
- Language :
- English
- ISSN :
- 1943-4936
- Volume :
- 31
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
- Publication Type :
- Academic Journal
- Accession number :
- 31378162
- Full Text :
- https://doi.org/10.1177/1040638719867743