The bisulfite modified DNA was then suspended in 20 μl of deionized water and used immediately or stored at -80°C until use. Bisulfite-specific (BSP) PCR and DNA sequencing The primers used to detect methylation of the SPARC gene promoter TRR were designed to specifically amplify bisulfite-converted DNA of SPARC TRR. The primers were 5′-ATTTAGTTTAGAGTTTTG-3′ (forward) and 5′-ACAAAACTTCCCTCCCTTAC-3′ (reverse) and were custom synthesized by Shanghai Sangon (Shanghai, China). Two microliters of the bisulfite modified DNA from each sample were subjected to PCR analysis in a 25 μL volume containing 1 × PCR buffer, 2.0 mmol/L MgCl2, 2.5 mmol/L dNTP, 1 mmol/L primer,

and EX Taq DNA INK1197 supplier HS 800 U/L. The reaction mixture was preheated at 95°C for

5 min and amplified using a touch-down PCR program (i.e., 9 cycles of 95°C for 30 s, 59°C for 30 s (next cycle touch-down 0.5°C) and 72°C for 30 s; 42 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension of 4 min at 72°C. The PCR products were then subjected to either direct sequencing analysis or cloning into the pMD-18-T vector (TaKaRa, Dalian, China) followed by sequencing analysis (after the cloning, 10-25 clones from each sample were randomly selected for DNA sequencing). Sequencing data analysis Sequencing analysis was performed by Shanghai Invitrogen Biotech Co. Ltd (Shanghai, China). For the data obtained from BSP PCR-based sequencing analysis, the A 1155463 percentage selleck chemicals of methylation of each CpG site in a given sample was calculated as the height of the “”C”" peak divided by the sum of the height of “”C”" + “”T”". Farnesyltransferase For the data obtained from BSP cloning-based sequencing analysis, the percentage

of methylation of each CpG site in a given sample was calculated as the number of the methylated CpG sites divided by the total observed sequenced clone numbers. The percentage of the region methylation in a given sample was the average of each CpG site in the DNA region. Statistical analysis Statistical analyses were conducted using SPSS version 15.0 (SPSS, Chicago, IL, USA). A one-way ANOVA test was performed to analyze differences in the percentage of the region methylation among pancreatic cancer tissues, adjacent normal pancreatic tissues, chronic pancreatitis tissues, and normal pancreatic tissues. General linear model univariate analysis was performed to determine the correlations of SPARC methylation with clinical characteristics of pancreatic cancer. All variables were subsequently analyzed using a stepwise multiple regression to assess their independent contribution to the methylation level, with entry and removal at the 0.05 and 0.1 significance levels, respectively.