Computational protocol: The influence of cytokine gene polymorphisms on the risk of developing gastric cancer in patients with Helicobacter pylori infection

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[…] Genomic DNA was extracted from the whole blood samples with EDTA using automated system for DNA isolation Magna Pure Compact Nucleic Acid Isolation Kit I (Roche Applied Science, Germany) on fully automated platform MagNa Pure Compact System in accordance to the instructions by the manufacturer. Complete nucleotide sequences of individual genes for inflammatory cytokine IL-1β (rs16944), TNF-α (rs1800629) and TLR-4 (rs4986790) were looked into online databases National Center for Biotechnology Information (NCBI; and ENSEMBLE ( The sequences were examined with the help of the software package Vector NTI Advance 11 (Invitrogen, Carlsbad, CA, USA).,, Polymorphisms genotyping was performed using the KASP technology (KBioscience competitive Allele-Specific PCR) using primers and reagents Kasp On Demand (KOD) (KBioscience, UK). 120 bp long reference sequences were sent to the manufacturer, upon which the appropriate primers and probes were designed ().The amplification of genomic DNA and the detection of polymorphisms were performed using the real-time polymerase chain reaction (PCR) apparatus LightCycler 480II (Roche Diagnostics GmbH, Germany). A touchdown protocol provided by the manufacturer was used: 94 °C for 15 min; 10 cycles of 94 °C for 10 s, 61 °C for 60 s (the anniling temperature dropped 0.6 °C per cycle to reach the annealing temperature of 55 °C) then; 26 cycles of 94 °C for 10 s, 55 °C for 60 s. IL-1RN gene contains a variable number of 86 base pair long tandem repeats (VNTR). Genomic DNA was amplified and PCR products were separated by the 1.5% agarose gel electrophoresis. Primers to detect IL-1RN*2/2 (TIB Molbiol, Germany) were used. We have used forward primer: 5′-CCCCTCAGCAACACTCC-3′, reverse primer: 5′-GGTCAGAAGGGCAGAGA-3′. Cycling conditions for the PCR were 95 °C for 15 min; 30 cycles of 94 °C for 30 s and 61 °C for 30 s; 72 °C for 60 s and 15 min at 72 °C. PCR reaction with the final volume of 25 μl was used, containing 12.5 μl of twice the reaction mixture of HotStartTaq Plus, 0.75 μl of each primer with a concentration of 10 μM, 8.5 μl of ddH2O and 2.5 μl of sample DNA.There are 5 versions of alleles. Allele 1, 2, 3, 4 and 5 carries 4, 2, 5, 3 and 6 repeats, respectively., Due to easier statistical analysis the allele polymorphisms were divided into short and long, the short allele being allele 2 and the long allele being those with 3 repeats or more (alleles 1, 3, 4, and 5). [...] The SPSS Statistics 21 (IBM, USA) software package was used for the statistical analysis. The Hardy-Weinberg equilibrium (HWE) of alleles in each individual locus was assessed. The degrees of freedom for HWE were calculated as the number of genotypes subtracted with the number of alleles. If the value of the c2 was less than 3.84, the frequencies of the population were in HWE. For all genotypes, the homozygote of the common allele was used as the reference. The IL-1B, IL-1RN, TNF-A and TLR-4 genotype frequencies for each polymorphism were compared by 2-sided Pearson c2 test, to evaluate the genotype distributions of categorical variables between each group of cases and controls, and to see if there was any association between the tested variables. The odds ratios (ORs) and the 95% confidence interval (95% CI) were assessed using logistic regression analysis with the reference category being healthy controls. ORs for different groups were adjusted for sex only. Statistical differences were considered to be significant at a P value < 0.05. […]

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