BisulFlash DNA Modification Kit

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	According to the predicted CpG islands and published literature, we developed one pair of nested MSP primers that is listed in Supplemental Table . Genomic DNA of ovarian cancer cells was extracted using a DNA Isolation Kit (Tiangen Biotech, Beijing, China). Bisulfite modification of DNA was performed following the recommended procedures of the BisulFlash DNA Modification Kit (Epigentek, Farmingdale, NY,USA). Placenta DNA was modified with the CpG Methyltransferase (M.SssI, NewEngland Biolabs, Ipswitch, MA, USA) and used as a positive control.

Protocol tips

According to the predicted CpG islands and published literature, we developed one pair of nested MSP primers that is listed in Supplemental Table . Genomic DNA of ovarian cancer cells was extracted using a DNA Isolation Kit (Tiangen Biotech, Beijing, China). Bisulfite modification of DNA was performed following the recommended procedures of the BisulFlash DNA Modification Kit (Epigentek, Farmingdale, NY,USA). Placenta DNA was modified with the CpG Methyltransferase (M.SssI, NewEngland Biolabs, Ipswitch, MA, USA) and used as a positive control.

Publication protocol

Bisulfite modification and methylation-specific PCR (MSP)
To predict CpG islands in the TIMP2 promoter, we searched the TIMP2 genomic sequence including two kb upstream and one kb downstream of the transcriptional start site (TSS) using the NCBI website (). The sequences were assessed with DataBase of CpG islands and Analytical Tool (DBCAT, ) according to the following criteria: the length of the CpG island >200 bp, observed/expected CpG ratio >0.6 and percentage of G plus C >50%. According to the predicted CpG islands and published literature, we developed one pair of nested MSP primers that is listed in Supplemental Table . Genomic DNA of ovarian cancer cells was extracted using a DNA Isolation Kit (Tiangen Biotech, Beijing, China). Bisulfite modification of DNA was performed following the recommended procedures of the BisulFlash DNA Modification Kit (Epigentek, Farmingdale, NY,USA). Placenta DNA was modified with the CpG Methyltransferase (M.SssI, NewEngland Biolabs, Ipswitch, MA, USA) and used as a positive control. Every 300 ng of genomic DNA was bisulfite-treated and then quantified with a NanoDrop 2000/2000C system (Thermo Scientific, USA). Modified DNA was first amplified with flanking primers followed by specific methylated or unmethylated primers using PrimeScript™ RT Master Mix (TaKaRa, Otsu, Japan). MSP products were subjected to electrophoresis on 3% agarose gels that were stained with ethidium bromide, detected and analyzed with Image Lab Software.

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