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Pipeline publication

[…] en selected for further methylation analyses. Genomic DNA was extracted from fresh frozen tissue (5 normal colon, 5 tumor samples) and HT-29 cells using High Pure PCR Template Preparation Kit (Roche). For calibration of the analysis, 0%, 2%, 25%, 50%, 75% and 100% artificially methylated DNA samples were prepared by mixing methylated (Universal Methylated Human DNA Standard, Zymo Research) and unmethylated (Unmethlyated EpiTect Control DNA, Qiagen) samples in the appropriate ratios. From each template 300 ng DNA was bisulfite converted by methylSEQr kit (Applied Biosystems) according to the manufacturer’s instructions. The CpG islands in the gene’s promoter region were predicted by CpG Plot EMBOSS Application http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.html and primers were designed using MethPrimer software http://www.urogene.org/methprimer/index1.html to amplify a region of the identified CpG islands. The specificity of the primers were tested in silico by the BiSearch software http://bisearch.enzim.hu (). Bisulfite-specific PCR reactions were perfomed in a final volume of 15 µl using AmpliTaq Gold 360 PCR Master Mix (2x) (Applied Biosystems), ResoLight HRM Dye (20x) (Roche), bisulfite-specific primers (200 nM) and 5 ng/well bisulfite converted DNA template. The amplification was carried out with the following thermocycling conditions: 95°C for 10 minutes, 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds for 10 touchdown cycles, 95°C for 30 seconds, 53°C for 30 seconds and 72°C for 30 seconds in 40 cycles. On completion of the PCR thermal cycling, for the HRM analysis the samples were denatured at 95°C for 1 minute, cooled down to 40°C and held for 1 minute, […]

Pipeline specifications

Software tools EMBOSS, methPrimer, BiSearch