High-throughput hacking of the methylation patterns in breast cancer by in vitro transcription and thymidine-specific cleavage mass array on MALDI-TOF silico-chip.
Over the last decade, the rapidly expanding interest in the involvement of DNA methylation in developmental mechanisms, human diseases, and malignancies has highlighted the need for an accurate, quantitative, and high-throughput assay. Existing methods are limited and are often too laborious for high-throughput analysis or inadequate for quantitative analysis of methylation. Recently, a MassCLEAVE assay has been developed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to analyze base-specific methylation patterns after bisulfite conversion. To find an efficient and more cost-effective high-throughput method for analyzing the methylation profile in breast cancer, we developed a method that allows for the simultaneous detection of multiple target CpG residues by using thymidine-specific cleavage mass array on matrix-assisted laser desorption/ionization time-of-flight silicon chips. We used this novel quantitative approach for the analysis of DNA methylation patterns of four tumor suppressor genes in 96 breast tissue samples from 48 patients with breast cancer. Each individual contributed a breast cancer specimen and corresponding adjacent normal tissue. We evaluated the accuracy of the approach and implemented critical improvements in experimental design.
Radpour
Ramin
R
Laboratory for Prenatal Medicine and Gynecologic Oncology, Women's Hospital/Department of Biomedicine, University of Basel, Basel, Switzerland.
Haghighi
Mahdi Montazer
MM
Fan
Alex Xiu-Cheng
AX
Torbati
Peyman Mohammadi
PM
Hahn
Sinuhe
S
Holzgreve
Wolfgang
W
Zhong
Xiao Yan
XY
D001483:Base Sequence; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000235:genetics*; D019175:DNA Methylation; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013936:Thymidine / Q000378:metabolism; D014158:Transcription, Genetic
D014408:Biomarkers, Tumor; D013936:Thymidine