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Pipeline publication

[…] µg/mL proteinase K (Boehringer Mannheim) at 48°C overnight, followed by phenol/chloroform extraction and ethanol precipitation of DNA as previously described., DNA was subjected to bisulfite treatment. Briefly, 1–2 µg of genomic DNA from HNSCC tissues and normal mucosa tissues were subjected to bisulfite treatment using the EpiTect® Bisulfite Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. This bisulfite treated DNA was then stored at −80°C., Bisulfite sequence analysis was performed to verify the methylation status in primary HNSCC tumors and non smoking normal mucosa tissues (UPPP). Bisulfite-treated DNA was amplified using bisulfite sequencing primers designed by MethPrimer to span areas of CpG islands in the promoter or first exon . Primer sequences were designed to not contain CG dinucleotides. Detailed primer sequences and PCR conditions are available upon request. The PCR products were gel-purified using the QIAquick 96 PCR Purification Kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. The purified PCR products were sequenced with both forward and reverse primers by GENEWIZ™., 1 ug of RNA was then utilized for cDNA synthesis. Reverse transcription was carried out using SuperScript First- Strand Synthesis kit (Invitrogen) and cDNA High Capacity Kit (Applied Biosystems). The final cDNA products were used as the templates for subsequent RT-PCR., RT PCR was performed either with SYBR Green technology (Quantifast SYBR Green PCR Kit (Qiagen, Valencia, CA)) using primer sets () that were designed using Primer3 and Integrated DNA techniques ( or with readymade primers-probe mix from Applied Biosystems (Assay Hs00427156_m1, Assay Hs03928990_g1). 18 s rRNA was examined to ensure accurate relative quantification in qRT-PCR. Serial dilutions of cell line cDNA, showing to express the specific gene studied, were used as positive controls to create standard curves. Each experiment was performed in triplicate using the ABI 7900HT real-time PCR machine. Expression values are presented as gene of interest (GOI)/18SX100., To selectively amplify demethylated promoter regions in genes of interest, probe and primers were designed using data from bisulfite sequencing of primary tumors which are […]

Pipeline specifications

Software tools methPrimer, GeneWiz, Primer3