Evaluation of a DNA Extraction and Purification Protocol Using Archived Formalin-fixed Paraffin-embedded Tissues for BRAF Mutations Analysis in Papillary Thyroid Microcarcinomas.
Author(s): Nechifor-Boilă A, Loghin A, Descotes F, Decaussin-Petrucci M, Borda A
Publication: Appl Immunohistochem Mol Morphol, 2019, Vol. 27, Page 70-76
PubMed ID: 28549037 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of formalin-fixed, paraffin-embedded (FFPE) block storage duration (1, 2, 3, or 4 years) and papillary thyroid tumor size on DNA concentration and purity and BRAF gene mutation detection by real-time RT-PCR. Differences in tumor size, block storage duration, DNA concentration, and DNA purity between BRAF-negative and BRAF-positive tumor specimens were also assessed.
Conclusion of Paper
DNA concentration, DNA purity, GAPDH crossing point (Cp) values, and BRAF gene Cp values were comparable between specimens from tumors ≤5 mm and tumors >5 mm and were not affected by FFPE block storage duration or BRAF mutation status.
Studies
-
Study Purpose
This study investigated the effects of FFPE papillary thyroid tumor block storage duration (1, 2, 3, or 4 years) and tumor size on DNA concentration and purity and BRAF gene mutation detection by real-time RT-PCR. Differences in tumor size, block storage duration, DNA concentration, and DNA purity between BRAF-negative and BRAF-positive tumor specimens were also assessed. Specimens from 25 patients with papillary thyroid microcarcinomas (PTMCs) undergoing surgery were immediately fixed in neutral buffered formalin for 48-72 hours (depending on the size of the specimen), paraffin embedded, and stored as blocks at room temperature for 1, 2, 3, or 4 years. Five 4-µm thick sections were obtained from each block, deparaffinized using xylene, and DNA was extracted using a MasterPure DNA Purification Kit. DNA yield (A260 nm) and purity (A260 nm/A280 nm) were assessed by spectrophotometry. The 15th exon of the BRAF gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified by real-time RT-PCR. The Cp was defined as the PCR cycle at which fluorescence was first detected. BRAF gene mutation status was assessed by high-resolution melting analysis and confirmed by Sanger sequencing.
Summary of Findings:
DNA concentration, DNA purity, GAPDH Cp values, and BRAF gene Cp values were comparable between specimens from tumors ≤5 mm and tumors >5 mm (P=0.400, P=0.470, P=0.520, and P=0.460; respectively) and were not affected by FFPE block storage duration (P=0.380, P=0.500, P=0.140, and P=0.300; respectively). BRAF mutation status was successfully determined in 24/25 cases and there were no significant differences between the BRAF-negative (n=16) and BRAF-positive (n=8) tumor specimens in tumor size, tumor block storage duration, DNA concentration, or DNA purity (P=0.693, P=0.282, P=0.243, and P=0.458; respectively).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA DNA sequencing DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 year
2 years
3 years
4 years
Real-time qPCR Specific Targeted nucleic acid BRAF
GAPDH
Preaquisition Prognostic factor ≤5 mm tumor
>5 mm tumor
BRAF-positive tumor
BRAF-negative tumor