Lls were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 fetal bovine serum. All cell lines were cultured in an atmosphere of 5 carbon dioxide at 37 .5-Aza-2-deoxycytidine treatmentspectrophotometric analysis were used to check RNA quality and quantity. Total RNA (5 g) was used to synthesize first-strand complementary DNA (cDNA) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The reaction mixture was diluted to 100 l with water, and 2.5 l of diluted cDNA mixture was added to each 25 l PCR reaction. The ZNF331 PCR primer sequences were as follows: 5-TAGGTCA GCTCTAGCCTCTC-3 (forward) and 5-AGCGTACC TTCACATATCCAG-3 (reverse). Thermal cycling parameters were as follows: 95 5 min; (95 30 s, 58 30 s, and 72 30 s) 35 cycles; 72 5 min. The primers for GAPDH were as follows: 5-GAGTCAACG GATTTGGTCGT-3 (forward), and 5-GACAAGCTT CCCGTTCTCAG-3 (reverse). Thermal cycling parameters were as follows: 95 5 min; (95 30 s, 58 30 s and 72 40 s) 25 cycles; 72 5 min. The amplified PCR products were examined by 1.5 agarose gels. Each cDNA sample was analyzed in triplicate with the Applied Biosystems StepOnePlus Real-Time PCR Systems using SYBR Green Realtime PCR Master Mix (Toyobo, Shanghai, China) according to the manufacturer’s instructions. The relative amount of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26740125 ZNF331 mRNA was normalized to GAPDH using the Ct method.Bisulfite modification, methylation-specific PCR, bisulfite sequencing, and KRAS and BRAF mutation detectionCRC cell lines (DLD1, SW48, HCT116, SW480, SW620, and DKO) were split to a low density (30 confluence) 12 h before treatment with 2 M 5-aza-2-deoxycytidine (DAC, Sigma, MO, USA). Growth medium conditioned with DAC at 2 M was exchanged every 24 h for a total of 96 h. At the end of the treatment course, RNA was extracted from the cells as described below.RNA isolation, semi-quantitative RT-PCR, and real-time quantitative RT-PCR analysesTotal RNA was isolated by Trizol reagent (Life Technologies, MD, USA). Agarose gel electrophoresis andGenomic DNA was extracted by the proteinase K method. Cultured cells and fresh tissue samples were digested by DNA digestion buffer (pH 8.0, 10 mM Luteolin 7-glucoside msds TrisCl, 25 mM EDTA, 1 SDS, 100 g/ml proteinase K) and extracted by phenol/chloroform. The bisulfite modification assay was performed as previously described [16]. MSP primers were designed according to genomic sequences around the transcription start sites (TSS) and synthesized (BGI, Beijing, China) to detect unmethylated (U) and methylated (M) alleles. The MSP primers were as follows: 5-TAAGGTAGGACGTTTTTAGGGTCGC3 (MF) and 5-AACTCTACACGACGCAAATAAAAC CG-3 (MR); 5-TTTTAAGGTAGGATGTTTTTAGGG TTGT-3 (UF) and 5-ACAACTCTACACAACACAAA TAAAACCA-3 (UR). The thermal cycling parameters were as follows: 95 5 min; (95 30 s, 60 30 s and 72 40 s) 35 cycles; 72 5 min. The expected sizes of unmethylated and methylated products were 147 and 142 bp, respectively. Bisulfite-treated DNA was also amplified using bisulfite sequencing (BSSQ) primers that included the MSP region. The sequencing primers were as follows: 5-GGTTATGAGTTATATTTTTTAGAAG3 (forward) and 5-CTCRCTCCTCATTAAACTATAC3 (reverse). The thermal cycling parameters were as follows: 95 5 min; (95 30 s, 55 30 s and 72 40 s) 35 cycles; 72 5 min. Methylation status was detected by MSP in four genes (RUNX3, CACNA1G, IGF2,Wang et al. Clinical Epigenetics (2017) 9:Page 3 ofand MLH1) to represent CpG island methylator phenotype (CIMP) according.