Bisulfite sequencing PCR

Genomic DNA was extracted from the harvested tumor metastases of the ovarian cancer mouse xenografts and SK-OV-3 cells using the QIAmp DNA mini kit (Qiagen) according to the manufacturer's protocol. Bisulfite treatment of the genomic DNA was performed using the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. For bisulfite sequencing of the target promoter region of GABRP, bisulfite sequencing PCR (BSP) was carried out using conventional PCR in a 50 μl reaction mixture containing 10 ng bisulfite-modified genomic DNA, 1.5 mM MgCl₂, 200 μM dNTP, 1 U Platinum Taq polymerase (Invitrogen), 1 × Platinum Taq buffer and 200 nM gene-specific BSP forward and reverse primers. The BSP primers were designed using MethPrimer software (http://www.urogene.org/methprimer). For GABRP, the BSP product was 452 bp (position in the human GRCh37/hg19 assembly: chromosome 5, 170209520–170209971) and contained five CpG sites. The five CpG sites are located at −1178, −1142, −994, −963 and −924 from the transcription start site. The BSP primer sequences were: (forward), 5′-TGAGTTTTTTTAGGAGAAATGAAAG-3′ and (reverse), 5′-CTAAACTCTAAATCATCCCCTCATC-3'. The reaction ran at 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, and a final elongation step at 72 °C for 5 min.

The BSP products were purified using the QIAquick Gel Extraction kit (Qiagen) according to the manufacturer's protocols and ligated into the yT&A cloning vector (Yeastern Biotech, Taipei, Taiwan). The ligation products were used to transform competent DH5α Escherichia coli cells (RBC Bioscience, Taipei, Taiwan) using standard procedures. Blue/white screening was used to select bacterial clones, and BSP product-positive clones were confirmed by colony PCR using the BSP primers to verify the insert size. Plasmid DNA was then extracted from at least 15 insert-positive clones using the QIAprep Spin Miniprep kit (Qiagen) and sequenced using the M13 primer to analyze the methylation status at specific CpG sites.