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IDENTIFICATION OF -308 TNF-α PROMOTER SINGLE NUCLEOTIDE POLYMORPHISM IN ACTIVE AND PASSIVE SMOKERS Gracia A.V. Pollo; Rooije R.H. Rumende; Trina E. Tallei
JURNAL ILMIAH SAINS Volume 19 Nomor 1, April 2019
Publisher : Sam Ratulangi University

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (656.16 KB) | DOI: 10.35799/jis.19.1.2019.22623

Abstract

IDENTIFICATION OF -308 TNF-α PROMOTER SINGLE NUCLEOTIDE POLYMORPHISM IN ACTIVE AND PASSIVE SMOKERS ABSTRACTTumour necrosis factor-α (TNF-α) is one of the pro-inflammatory cytokines that play a role in the inflammatory process, immune system development, and apoptosis. This protein is encoded by the TNF-α gene. Several studies had linked the presence of polymorphisms in the TNF-α promoter region with susceptibility to the onset of chronic obstructive pulmonary disease (COPD) in active and passive smokers. This study aimed to identify and analyze the nucleotide polymorphism in TNF-α gene promoter regions in active and passive smokers in Manado. The method used in this study included blood sampling, DNA extraction, amplification of -308 TNF-α promoter region, sequencing and data analysis. The softwares used for data analysis were Geneious, Multalin, Clustal Omega and DnaSP. DnaSP was used to compute the level of polymorphism based on haplotype statistics. The results did not show single nucleotide polymorphism at position -308 in TNF-α gene promoters in active and passive smokers. This was because at that position, the nucleotides were both in the form of guanosine monophosphate and there were no mutation caused by cigarette smoke exposure.Keywords: active smoker, passive smoker, single nucleotide polymorphism, TNF-α promoter DETEKSI POLIMORFISME NUKLEOTIDA TUNGGAL-308 PROMOTERTNF-α PADA PEROKOK AKTIF DAN PASIF ABSTRAKTumor necrosis factor-α (TNF-α) merupakan salah satu sitokin pro-inflamasi yang berperan dalam proses inflamasi, perkembangan sistem imun, dan apoptosis. Protein ini dikode oleh gen TNF-α. Beberapa penelitian telah mengkaitkan adanya polimorfisme pada daerah promoter TNF-α dengan kerentanan terhadap timbulnya chronic obstructive pulmonary disease (COPD) pada perokok aktif maupun pasif. Penelitian ini bertujuan untuk mengidentifikasi dan menganalisis polimorfisme nukleotida tunggal pada posisi -308 dari promoter gen TNF-α perokok aktif dan pasif. Metode yang digunakan dalam penelitian ini meliputi pengambilan sampel darah, ekstraksi DNA, amplifikasi daerah -308 promoter gen TNF-α, sekuensing serta analisis data. Analisis data menggunakan perangkat lunak Geneious, BLAST, Multalin, Clustal Omega dan DnaSP. Perangkat DnaSP akan mengkomputasi tingkat polimorfisme berdasarkan statistik haplotype-based. Hasil yang didapatkan menunjukkan bahwa polimorfisme nukleotida tunggal pada posisi -308 dari promoter gen TNF-α antara perokok aktif dan pasif tidak ditemukan. Hal ini dikarenakan oleh pada posisi tersebut, nukleotidanya sama-sama berbentuk guanosin monofosfat dan tidak terjadi mutasi akibat pengaruh paparan asap rokok.Kata kunci: perokok aktif, perokok pasif, single nucleotide polymorphism, TNF-α promoter
In Silico and Validation Approaches for Optimum Conditions of Rattus norvegicus Target Gene qPCR Primers Gracia Alice Victoria Pollo; Nyoman Yudi Antara; Firman Alamsyah; Rarastoeti Pratiwi
Journal of Tropical Biodiversity and Biotechnology Vol 8, No 2 (2023): August
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/jtbb.71765

Abstract

The qPCR method requires an oligonucleotide pair to prime the amplification process. With the variety of qPCR reagent and primer options available, in silico and laboratory experimental validation approach was needed to validate the most suitable primer for prior use. This article aims to provide in silico analysis of actin alpha-2 smooth muscle (Acta2), fibroblast activation protein (Fap), hypoxanthine phosphoribosyltransferase-1 (Hprt1), platelet-derived growth factor subunit B (Pdgfb), phosphoinositide-3-kinase regulatory subunit-1 (Pik3r1), and vascular cell adhesion molecule-1 (Vcam1) qPCR primer with qPCR and electrophoresis validation. The procedure used in this approach was in silico analysis of primer from published articles and newly designed primer. The analysis was done with Primer-BLAST for gene specificity, Primer-Dimer, OligoCalc for hairpin formation, BLAST Nucleotide for identical sequence screening, and Clustal Omega for product length validation. Experimental validation was done using qPCR for optimal annealing temperature, priming ability, and amplificon specificity, and electrophoresis for product length validation. This assessment resulted in in silico and laboratory experimental validation of Acta2, Fap, Hprt1, Pdgfb, Pik3r1, and Vcam1 primer pairs producing suitable amplicon for qPCR using Rattus norvegicus cDNA with SYBR annealing temperature range of 60-65°C with three mM MgCl2. The primer pair can be used for further qPCR analysis under similar conditions and the procedure stated can be used as starting point for qPCR Primer preparation.