br R ACAACCCTCGCAACTCCG br OGDHL unmeth F TTAGGGTTTTGTGTAGAG
OGDHL(unmeth) F:5-TTAGGGTTTTGTGTAGAGGAGTG-3 61 114
radiotherapy and have no history of other diagnosed malignancies. Tumor and matched tumor free margin samples were obtained from 40 patients. Tissue samples were validated as being tu-mor and tumor free margin by standard pathology analysis and were referred to the laboratory under certain conditions with complete patient information including clinicopathologic and de-mographic data. Following resection, the tissue samples were immediately snap-frozen in liquid nitrogen and stored at -80°C in the laboratory until further steps.
DNA extraction and sodium bisulfite modification
Total genomic DNA was extracted from tissue samples using previously described standard phenol chloroform method. DNA concentrations were measured using a NanoDrop spectropho-tometer, and then stored at -20°C until the next step. Extracted DNA samples were excluded from further analysis if the final concentration was <100 ng/μL, or the A260/A280 ratio was outside the range of 1.7-1.9. In the next step, total genomic DNA samples were subjected to sodium bisulfate modification using EZ DNA methylation-Gold kit (Zymo Research Corp. Irvine, CA), according to the instructions provided by the manufacturer. The modified DNA (Bis-DNA) samples were stored immediately at -20°C.
Quantitative methylation-specific real time PCR
Methylation analysis was done using qMSP assay. Two sets of primer pairs (methylated and unmethylated) were designed, considering the qMSP primer design guidelines, for specific GpC rich islands in Oxaliplatin region of OGDHL gene (Table 1). For each reaction, 2 μL of Bis-DNA tem-plate, 10 μL of master mix (SYBR Premix Ex Taq II), 1 μL of specific primers mix, and 7 μL of dou-ble distilled water were added. Step one plus Real Time PCR instrument (Applied bio systems) was used and the assays were conducted with the following conditions: initial denaturation at 95°C for 5 minutes; and then 45 cycles at 95°C of denaturation for 20 seconds, appropriate an-nealing temperature for each primer set (Table 1) for 15 seconds, and extension at 72°C for 20 seconds. After 45 cycles 10 minutes final extension was applied at 72°C. Each sample was run as duplicates for analysis. We calculate the percent of methylation for paired tumor and mar-gin samples as previously descripted14: % meth = 100/[1+2 Ct (meth-unmeth)]%. Subtracting the Ct values of methylated OGDHL from unmethylated OGDHL resulted in Ct(meth-unmeth).
Descriptive tests such as mean ±SD, frequency, and median was applied. Statistical analysis was performed on obtained data using Mann-Whitney, Kruskal-Wallis, and Spearman correlation coe cient test for analyzing probable correlations of clinicopathologic manifestations and gene methylation. Sensitivity and specificity of methylation status as a biomarker for CRC were as-sessed by receiver operating characteristic (ROC) curve analysis. All tests performed using SPSS version 22 (SPSS Inc., Chicago, IL) and P < 0.05 was considered statistically significant for all tests.
Please cite this article as: M. Khalaj-kondori, M. Hosseinnejad and A. Hosseinzadeh et al., Aberrant hypermethyla-tion of OGDHL gene promoter in sporadic colorectal cancer, Current Problems in Cancer, https://doi.org/10.1016/j. currproblcancer.2019.03.001
ARTICLE IN PRESS
4 M. Khalaj-kondori, M. Hosseinnejad and A. Hosseinzadeh et al. / Current Problems in Cancer xxx (xxxx) xxx
Clinicopathologic features of 40 CRC patients involved in the study and correlation of these features with methylation of OGDHL gene promoter.