Hydroxyquinone

Genomic DNA of human cartilage (5 mg) was denatured by 2 M NaOH for 15 min at 50°C. 2% low-melting agarose were subsequently added to the DNA solution, and agarose beads were formed after pipetting 15-μl DNA/agarose mixture into cold mineral oil. The DNA/agarose beads were treated with freshly prepared hydroxyquinone (10 mM; Sigma) and sodium bisulfite (40.5%, pH 5; Sigma) at 50°C for 16 h under mineral oil. The reaction was stopped by 0.3 M NaOH for 10 min at room temperature.
The PCR amplifications were performed in 25 μl reactions containing one agarose/DNA bead. The following primer sequences were used: 5′-ATTAATATTATAGATAATT-3′ (forward), 5′-ATTATATATTTATTATTGTGT-3′ (reverse); The PCR products were separated by agarose gel, purified and cloned into the pMD18-T vector system (Takara). Fifteen clones of each sample were sequenced, and the sense strands were used to evaluate CpG site methylation status.

Five microgram genomic DNA of human SF was denatured by 2 M NaOH for 15 min at 50 °C. 2% low-melting agarose were subsequently added to the DNA solution, and agarose beads were formed after pipetting 15-μl DNA/agarose mixture into cold mineral oil. The DNA/agarose beads were treated by freshly prepared hydroxyquinone (10 mM; Sigma) and sodium bisulfite (40.5%, pH 5; Sigma) at 50 °C for 16 h under mineral oil. The reaction was stopped by 0.3 M NaOH for 10 min at room temperature.
The PCR amplifications were performed in 25 μl reactions containing one agarose/DNA bead. The following primer sequences were used: Region 1: 5′-TTTTTATATTAAAGAATTTT-3′ (forward), 5′-TTTATTTATTAAATATGGTGT-3′ (reverse); Region 2: 5′-TAGGTGAAGAAAGTGGTAGA-3′ (forward), 5′-GATTAGATTAATAGGTTAGAA-3′ (reverse) and Region 3: 5′-TTTTTAGTTTTGGAATTGTT-3′ (forward), 5′-AGGTAATATTAGGAGTAGTTTT-3′ (reverse). The PCR products were separated by agarose gel, purified and cloned into the pMD18-T vector system (Takara). Fifteen clones of each sample were sequenced, and the sense strands were used to evaluate CpG site methylation status.

Genomic DNA of human cartilage (5 mg) was denatured by 2 M NaOH for 15 min at 50°C. 2% low-melting agarose were subsequently added to the DNA solution, and agarose beads were formed after pipetting 15-μl DNA/agarose mixture into cold mineral oil. The DNA/agarose beads were treated with freshly prepared hydroxyquinone (10 mM; Sigma) and sodium bisulfite (40.5%, pH 5; Sigma) at 50°C for 16 h under mineral oil. The reaction was stopped by 0.3 M NaOH for 10 min at room temperature.
The PCR amplifications were performed in 25 μl reactions containing one agarose/DNA bead. The following primer sequences were used: 5′-ATTAATATTATAGATAATT-3′ (forward), 5′-ATTATATATTTATTATTGTGT-3′ (reverse); The PCR products were separated by agarose gel, purified and cloned into the pMD18-T vector system (Takara). Fifteen clones of each sample were sequenced, and the sense strands were used to evaluate CpG site methylation status.