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Immortalised human epithelial cells (human telomerase reverse transcriptase-human pancreatic nestin-expressing ductal cells) were supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on week 4, gDNA was extracted from the cells (3×10 4 cells) and bisulphite conversion of the gDNA (750 ng) was carried out using the MethylDetector Bisulfite Modification Kit. DNA was also extracted from HT-29 cells (methylated control) and HDBEC (unmethylated control) and processed as aforementioned. The modified DNA (10 ng/reaction) was amplified with methylation-specific, and unmethylated-specific sets primers to the p16 gene promoter region. Each of the nested amplification steps was carried out for 35 cycles at an annealing temperature of 60°C. Aliquots (4 µl) from the outer reactions were then used as the template for the inner PCR reactions using primers specific for methylated and unmethylated DNA. Both amplicons generated bands of 149 bp. A control β-actin sample was also amplified and examined alongside. (A) The products were examined by 2% (w/v) agarose gel electrophoresis and (B) the band intensities determined and the ratios of the methylated:unmethylated DNA calculated (n=3). TF, tissue factor; gDNA, genomic DNA.

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Immortalised human epithelial cells (human telomerase reverse transcriptase-human pancreatic nestin-expressing ductal cells) were supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on week 4, gDNA was extracted from the cells (3×10 4 cells) and bisulphite conversion of the gDNA (750 ng) was carried out using the MethylDetector Bisulfite Modification Kit. DNA was also extracted from HT-29 cells (methylated control) and HDBEC (unmethylated control) and processed as aforementioned. The modified DNA (10 ng/reaction) was amplified with methylation-specific, and unmethylated-specific sets primers to the p16 gene promoter region. Each of the nested amplification steps was carried out for 35 cycles at an annealing temperature of 60°C. Aliquots (4 µl) from the outer reactions were then used as the template for the inner PCR reactions using primers specific for methylated and unmethylated DNA. Both amplicons generated bands of 149 bp. A control β-actin sample was also amplified and examined alongside. (A) The products were examined by 2% (w/v) agarose gel electrophoresis and (B) the band intensities determined and the ratios of the methylated:unmethylated DNA calculated (n=3). TF, tissue factor; gDNA, genomic DNA.

Article Snippet: Bisulphite conversion of the gDNA (750 ng) was carried out using the MethylDetector Bisulfite Modification Kit (Active Motif, Inc.) according to manufacturer's instructions.

Techniques: Reverse Transcription, Expressing, Recombinant, Modification, Methylation, Control, Amplification, Generated, Agarose Gel Electrophoresis