Τίτλος:
A rapid and accurate closed-tube Methylation-Sensitive High Resolution Melting Analysis assay for the semi-quantitative determination of SOX17 promoter methylation in clinical samples
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
Introduction: SOX17 promoter methylation can provide important prognostic information in cancer. We developed a novel semi-quantitative MS-HRMA assay for SOX17 promoter methylation. Methods: The assay was optimized by using synthetic control samples and validated by analyzing 165 clinical samples: a) 107 formalin fixed paraffin embedded (FFPEs) samples of patients with early breast cancer, b) 27 FFPE samples of patients with metastatic breast cancer, c) 15 reduction mammoplasty specimens obtained from healthy women and d) 16 genomic DNA samples isolated from healthy blood donors. Comparison with real time MSP was also performed. Results: The assay is highly specific and sensitive and provides a semi-quantitative estimation of SOX17 promoter methylation. SOX17 promoter was found methylated in 96/134 (71.6%) breast cancer samples, while none of the 31 non-cancerous samples tested was positive (0%). SOX17 promoter methylation levels varied significantly among samples. When 165 clinical samples were analyzed both by MS-HRMA and real time MSP results were significantly comparable (concordance: 146/165, 88.5%). Conclusions: This novel MS-HRMA assay for SOX17 promoter methylation is closed-tube, highly sensitive, specific, cost-effective, rapid and easy-to-perform. It gives comparable results to Real-Time MSP in less time, while it offers the advantage of additionally providing an estimation of SOX17 promoter methylation levels. © 2015.
Συγγραφείς:
Mastoraki, S.
Chimonidou, M.
Dimitrakopoulos, L.
Kounelis, S.
Malamos, N.
Georgoulias, V.
Lianidou, E.
Περιοδικό:
Clinica Chimica Acta
Λέξεις-κλειδιά:
genomic DNA; transcription factor Sox17; SOX17 protein, human; transcription factor Sox, Article; breast cancer; breast metastasis; closed tube methylation sensitive high resolution melting analysis; computer model; controlled study; cost effectiveness analysis; diagnostic accuracy; diagnostic test accuracy study; DNA methylation; early cancer; female; high resolution melting analysis; human; human tissue; intermethod comparison; major clinical study; polymerase chain reaction system; priority journal; promoter region; protein methylation; real time methylation specific polymerase chain reaction; reproducibility; sensitivity and specificity; SOX17 gene; temperature; breast tumor; DNA denaturation; genetics; promoter region; transition temperature; validation study, Breast Neoplasms; DNA Methylation; Female; Humans; Nucleic Acid Denaturation; Promoter Regions, Genetic; SOXF Transcription Factors; Transition Temperature
DOI:
10.1016/j.cca.2015.02.035
