Computational protocol: Association of interleukin 1 beta (IL-1β) polymorphism with mRNA expression and risk of non small cell lung cancer☆

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[…] DNA extraction was performed according to the manufacturer's protocol for Qiagen DNA extraction kits (Qiagen, Hilden, NRW, Germany). DNA content was quantified by spectrophotometric absorption (Nanodrop Spectrophotometer, BioLab, Scoresby, VIC, Australia). Polymerase chain reaction (PCR) was performed using an iCycler Thermal Cycler (Bio- Rad, Hercules, CA, USA). Interleukin 1 beta-31C > T (rs1143627) and interleukin 1 beta-511 T > C (rs16944) genotypes were determined using polymerase chain reaction–restriction fragement length polymorphism (PCR-RFLP) method followed by DNA sequencing. Amplification of the target region was carried out by polymerase chain reaction using the specific forward and reverse primers. Primers were designed and selected using Primer3, version 0.4.0 software. For interleukin 1 beta-31 C > T, the primers were forward 5′-AGAAGCTTCCACCAATACTC-3′ and reverse 5′-AGCACCTAGTTGTAAGGAAG-3′. For interleukin 1 beta-511 T > C, the primers were forward 5′-TGG CAT TGA TCT GGT TCA TC-3′and reverse 5′-GTT TAG GAA TCT TCC CAC TT-3′. The PCR reaction mixture consisted of Taq 1.5 U (Ferments), sense and antisense primers (0.5 μmol/l), MgCl2 (50 mmol/l), dNTP (0.2 mmol/l), and DNA template (1 μg) and was subjected to an initial denaturing step of 4 min at 95 °C, then 35 cycles of denaturing for 30 s at 95 °C, annealing for 30 s at 56 °C, extension for 30 s at 72 °C, and a final extension step of 10 min at 72 °C. Digestion of the amplified products of interleukin 1 beta-31C > T and interleukin 1 beta-511 T > C was done by using 10 units restriction endonucleases Alu1 (New England Biolabs) and Ava1 (New England Biolabs) respectively and incubated at 37 °C for 16 h. The digested products were checked on 3% agaroses gel, the RFLP picture for iinterleukin 1beta − 31 genotype was identified as (C/C - 239 bp), (T/T- 137/102 bp), (T/C- 239/137/102 bp) and interleukin 1 beta-511 was identified as (T/T- 304 bp), (C/C -190/114), (T/C- 304/190/114 bp) . RFLP results were later confirmed by DNA sequencing . [...] Total RNA was extracted by using TRIZOL (Sigma Aldrich, USA) from normal tissues of lung cancer tissue. Integrity of the mRNA was checked on 1% agarose gel and quantified at 260/280 ratio. RNA was converted to cDNA by using first strand cDNA synthesis kit according to manufactures protocol (Fermentas, USA). Dilution of the cDNA was performed to get uniform quantity of cDNA in all samples. PCR primers were designed for interleukin 1 beta and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) over introns to avoid the amplification of possible traces of genomic DNA contamination by using Primer Express 3.0 software, Applied Biosystems and glyceraldehyde 3-phosphate dehydrogenase mRNA (GenBank accession number NM-002046). Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. For interleukin 1 beta primer sequence were as follows- forward 5'-GCACGATGCACCTGTACGAT and reverse 5'-CACCAAGCTTTTTTGCTGTGAGT-3' and for glyceraldehyde 3-phosphate dehydrogenase, the primers were as follows: forward 5′-GATCCGCATAATCTGCATGGT-3′ and reverse 5′-GATCCGCATAATCTGCATGGT-3′. Quantitative Real time PCR (Agilent Biotechnologies, Germany) was performed for the detection of interleukin 1 beta mRNA by recruiting Appllied Biosystems Inc StepOne software v2.0. PCR was performed containing Maxima® SYBR Green qPCR Master Mix (2X) and all the samples (unknown and standards) were run in triplicates and accompanied by non template control (NTC). Thermal cycling conditions included 40 cycles of 30s at 55 °C. The melting curves of all final real time PCR products were analysed for determination of genuine products and contamination of non specific products and primer dimer. All amplified products of real time polymerase chain reaction were subjected for separation on 2% agarose gel electrophoresis for ensuring the correct amplification products. Delta CT (ΔCT) method was used to check the interleukin 1 beta mRNA expression in normal lung tissue of lung cancer patients by normalization against glyceraldehyde 3-phosphate dehydrogenase used as a reference gene. […]

Pipeline specifications

Software tools Primer3, Primer Express
Application qPCR
Organisms Homo sapiens
Diseases Carcinoma, Non-Small-Cell Lung, Lung Neoplasms