CR and PR were considered to be a good response; SD and PD, a poor response. DNA extraction Genomic DNA was extracted from peripheral blood lymphocytes by the routine phenol/chloroform method. First, white blood cells were separated from red blood cells by washing three times in phosphate buffer solution. Then, the DNA was extracted with phenol/chloroform and was precipitated with cold

ethanol. All DNA samples were dissolved in water and stored at -20°C. Genotyping The two SNPs were detected using modified polymerase chain reaction (PCR) mismatch amplification (MA-PCR). The two forward primers for XRCC1 gene Arg194Trp site were 5′-GGGGGCTCTCTTCTTCAGGC-3′ and 5′-GGGGGCTCTCTTCTTCAGGT-3′, which differ in the last base; the reverse primer selleck compound was 5′-CGCTGGCTGTGACTATGAAG-3′, which together produce a 362 bp fragment. The two forward primers for the XRCC1 gene Arg399Gln site were 5′-CGTCGGCGGCTGCCCTCCTG-3′ and 5′-CGTCGGCGGCTGCCCTCCTA-3′; the reverse primer was 5′-TTACAGGCGTGAGCCACTGC-3′, which together produce a 354 bp fragment. For assessing the reproducibility of results, all samples were tested twice by different technical personnel and the CFTRinh-172 chemical structure results were concordant for all masked duplicate sets. Detection of protein expression Primary Antibodies

The rabbit anti-human polyclonal antibodies specific for XRCC1 were purchased from Santa Cruz Biotechnology™, Inc, Santa Cruz, California,

USA. Immunohistochemistry 3-MA price and Evaluation XRCC1 protein expression was detected by Immunohistochemistry, using the EnVision two-step method. The cervical carcinoma samples from patients were obtained from the paraffin-embedded tissue blocks from cervical biopsy before therapy. The quantitative immunoreactive Hydroxychloroquine scores (H-Score method) were used to evaluate the results, calculated by Σp(i+1), with i representing the various levels of stain: 0, no detectable stain in the nucleus or cytoplasm; 1, yellowish stain; 2, yellow stain; 3, brown stain; p represented the percentage of samples of each stain level. Five random fields (400× objective) were counted, and slides were reviewed independently by two pathologists without knowledge of the clinical data, The average of the the quantitative immunohistochemical scores data was calculated as the final result for each sample. Statistical analysis Difference in frequencies of the XRCC1 genotypes and alleles between the different chemotherapy response groups were evaluated by X 2 test and Fisher’s test. The association between XRCC1 polymorphisms and protein expression were evaluated by variance analysis. We also evaluated the observed genotype frequencies with those calculated from the Hardy-Weinberg equilibrium equation (p2+2pq+q2 = 1, where p is the frequency of the variant allele and q = 1-p).