Mers used for amplification of hsamiR-27a mRNA were 59- ACACTCCAGCTGGGTTCACAGTGGCTAAG-39 (forward) and 59TGGTGTCGTGGAGTCG-39 (reverse), and the primers for U6 were 59- CTCGCTTCGGCAGCACA-39 (forward) and 59AACGCTTCACGAATTTGCGT-39 (reverse). All reactions were conducted in triplicate. Fold changes were normalized to the expression levels 
of U6.Materials and Methods Study subjectsThis study comprised 594 patients and 600 cancer-free controls. All subjects in our study are ethnic Han Chinese with no genetic relationship. All the patients were newly diagnosed with histopathologically confirmed incident RCC. Those cases that received chemotherapy or radio-therapy before surgery or had other type of cancer were excluded from the present study. Consecutive RCC patients were recruited between May 2004 and August 2010 at The First Affiliated Hospital of Nanjing Medical University, Nanjing, China. Disease was classified according to World Health Organization criteria and staged according to the American Joint Committee on Cancer TNM classification. The Fuhrman scale was used to assess tumor nuclear grade [20]. The controls were recruited from those who were seeking health care in the outpatient departments at the same hospital. The cancer-free controls were frequency 58-49-1 chemical information matched by sex and age (65 years) to the cases without individual history of 25033180 cancer and family unrelated to the cases. A guided questionnaire on demographic and lifestyle factors was administered through face-to-face interviews by trained interviewers. Each patient donated 5 ml blood for genomic DNA extraction after a written informed consent obtaining from all subjects. This study was approved by the institutional review board of Nanjing Medical University. For the survival analysis, 296 RCC cases enrolled in our ongoing cohort study from May 2004 to October 2009 were used. The patients were followed up prospectively every 6 months from the date receiving a confirmed diagnosis until death or last time ofStatistical analysisDifferences in the distributions of selected demographic variables and frequencies of genotypes between the cases and controls were evaluated by using the Student’s t-test (for continuous variables) or Pearson’s x2-test (for categorical variables). Hardy-Weinberg equilibrium (HWE) of the controls’ genotype frequencies was assessed by a goodness-of-fit x2 test. The association between the SNP rs895819 polymorphism and RCC risk were estimated by computing odds ratios (ORs) and their 95 confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for possible confounders. The Kaplan-Meier method, log-rank test, univariate and multivariate Cox regression analyses were used to evaluate the effects of pre-miR-27a genotypes on the overall survival of patients with RCC. P,0.05 was considered statistically significant. All the statistical analyses were done with Statistical Analysis System software (9.1.3; SAS Institute, Cary, NC, U.S.) with two-sided P values. The statistical power was calculated by using the PS software (http://biostat.mc.vanderbilt.edu/twiki/bin/view/ Main/PowerSampleSize).pre-miR-27a Polymorphism and RCC RiskFigure 1. DNA sequencing chromatograms of three different samples of PCR products confirmed rs895819 polymorphism. (A) Double peaks labeled with an arrow represented the order BI 78D3 heterozygous genotype TC (AG). (B) Single peak labeled with an arrow represented the homozygous genotype TT (AA). (C) Single peak labeled with an.Mers used for amplification of hsamiR-27a mRNA were 59- ACACTCCAGCTGGGTTCACAGTGGCTAAG-39 (forward) and 59TGGTGTCGTGGAGTCG-39 (reverse), and the primers for U6 were 59- CTCGCTTCGGCAGCACA-39 (forward) and 59AACGCTTCACGAATTTGCGT-39 (reverse). All reactions were conducted in triplicate. Fold changes were normalized to the expression levels of U6.Materials and Methods Study subjectsThis study comprised 594 patients and 600 cancer-free controls. All subjects in our study are ethnic Han Chinese with no genetic relationship. All the patients were newly diagnosed with histopathologically confirmed incident RCC. Those cases that received chemotherapy or radio-therapy before surgery or had other type of cancer were excluded from the present study. Consecutive RCC patients were recruited between May 2004 and August 2010 at The First Affiliated Hospital of Nanjing Medical University, Nanjing, China. Disease was classified according to World Health Organization criteria and staged according to the American Joint Committee on Cancer TNM classification. The Fuhrman scale was used to assess tumor nuclear grade [20]. The controls were recruited from those who were seeking health care in the outpatient departments at the same hospital. The cancer-free controls were frequency matched by sex and age (65 years) to the cases without individual history of 25033180 cancer and family unrelated to the cases. A guided questionnaire on demographic and lifestyle factors was administered through face-to-face interviews by trained interviewers. Each patient donated 5 ml blood for genomic DNA extraction after a written informed consent obtaining from all 
subjects. This study was approved by the institutional review board of Nanjing Medical University. For the survival analysis, 296 RCC cases enrolled in our ongoing cohort study from May 2004 to October 2009 were used. The patients were followed up prospectively every 6 months from the date receiving a confirmed diagnosis until death or last time ofStatistical analysisDifferences in the distributions of selected demographic variables and frequencies of genotypes between the cases and controls were evaluated by using the Student’s t-test (for continuous variables) or Pearson’s x2-test (for categorical variables). Hardy-Weinberg equilibrium (HWE) of the controls’ genotype frequencies was assessed by a goodness-of-fit x2 test. The association between the SNP rs895819 polymorphism and RCC risk were estimated by computing odds ratios (ORs) and their 95 confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for possible confounders. The Kaplan-Meier method, log-rank test, univariate and multivariate Cox regression analyses were used to evaluate the effects of pre-miR-27a genotypes on the overall survival of patients with RCC. P,0.05 was considered statistically significant. All the statistical analyses were done with Statistical Analysis System software (9.1.3; SAS Institute, Cary, NC, U.S.) with two-sided P values. The statistical power was calculated by using the PS software (http://biostat.mc.vanderbilt.edu/twiki/bin/view/ Main/PowerSampleSize).pre-miR-27a Polymorphism and RCC RiskFigure 1. DNA sequencing chromatograms of three different samples of PCR products confirmed rs895819 polymorphism. (A) Double peaks labeled with an arrow represented the heterozygous genotype TC (AG). (B) Single peak labeled with an arrow represented the homozygous genotype TT (AA). (C) Single peak labeled with an.
