Evaluation of a DNA Extraction and Purification Protocol Using Archived Formalin-fixed Paraffin-embedded Tissues for BRAF Mutations Analysis in Papillary Thyroid Microcarcinomas
| Autor: | Adela Nechifor-Boilă, Andrada Loghin, Françoise Descotes, Angela Borda, Myriam Decaussin-Petrucci |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Proto-Oncogene Proteins B-raf
0301 basic medicine Histology Thyroid Gland Gene mutation Biology medicine.disease_cause Polymerase Chain Reaction Pathology and Forensic Medicine law.invention 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine law medicine Carcinoma Humans Thyroid Neoplasms Polymerase chain reaction Biological Specimen Banks Mutation Paraffin Embedding High-Throughput Nucleotide Sequencing DNA medicine.disease Molecular biology DNA extraction Carcinoma Papillary Housekeeping gene Medical Laboratory Technology genomic DNA 030104 developmental biology chemistry 030220 oncology & carcinogenesis |
| Zdroj: | Applied Immunohistochemistry & Molecular Morphology. 27:70-76 |
| ISSN: | 1541-2016 |
| DOI: | 10.1097/pai.0000000000000535 |
| Popis: | The isolation of good quality genomic DNA from formalin-fixed, paraffin-embedded tissues is challenging, especially in cases of small tissue specimens. The aim of our study was to evaluate a DNA extraction protocol using formalin-fixed, paraffin-embedded tissues in our laboratory and apply this method to a series of papillary thyroid microcarcinomas (PTMCs). A total of 25 PTMCs and 3 papillary thyroid carcinoma control cases were included in the study. We assessed a DNA extraction protocol on the basis of a precipitation method (MasterPure DNA purification kit, Epicentre), according to the manufacturer’s instructions. All PTMCs were subject to real-time polymerase chain reaction (PCR) amplification targeting the BRAF gene and a housekeeping gene (GAPDH). BRAF gene mutations were then assessed by high-resolution melting analysis and confirmed by sequencing of the PCR products. Using this extraction method, we produced good yields of DNA (mean concentration, 147.4±77.8 ng/µL), in addition to high levels of purity (mean A260/A280 ratio: 1.63±0.1). We successfully assessed the BRAF mutation status in 24 cases (16 BRAF-negative; 8 BRAFV600E positive), although 1 case revealed an inconclusive pattern following high-resolution melting analysis and sequencing of the PCR products. We observed no differences in the tumor size (P=0.693), storage period of the tumor block (P=0.282), DNA concentration (P=0.243), DNA purity (P=0.458), CpGAPDH (P=0.173), or CpBRAF (P=0.217) values between the BRAF-mutated and nonmutated group of PTMCs. Our findings demonstrate the importance of a reliable, reproducible DNA extraction technique for efficient PCR amplification, uniformly applied to all cases in this study, regardless of the BRAF mutation status. |
| Databáze: | OpenAIRE |
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