Development and Evaluation of a SYBR Green Real-time PCR Assay for Canine Cytokine Gene Expression

SYBR Green 실시간 역전사 중합효소연쇄반응을 이용한 개 싸이토카인 유전자 발현의 정량

  • Yu, Do-Hyeon (College of Veterinary Medicine & Bio-Safety Research Institute, Chonbuk National University) ;
  • Ihn, Dong-Chul (College of Veterinary Medicine & Bio-Safety Research Institute, Chonbuk National University) ;
  • Park, Chul (College of Veterinary Medicine & Bio-Safety Research Institute, Chonbuk National University) ;
  • Park, Jin-Ho (College of Veterinary Medicine & Bio-Safety Research Institute, Chonbuk National University)
  • 유도현 (전북대학교 수의과대학) ;
  • 인동철 (전북대학교 수의과대학) ;
  • 박철 (전북대학교 수의과대학) ;
  • 박진호 (전북대학교 수의과대학)
  • Accepted : 2010.10.05
  • Published : 2010.10.30

Abstract

Cytokines are important mediators of the immune response, and quantitating cytokine mRNA is a highly sensitive and attractive method for measuring cytokine production. The objective of the current study was to develop and validate a SYBR green quantitative real-time reverse transcriptase PCR (qRT-PCR) assay for measuring canine cytokine mRNA. The optimal annealing temperatures ($T_a$) of the designed primers were $62^{\circ}C$ for interleukin (IL)-$1{\beta}$, IL-6 and IL-10; $60^{\circ}C$ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tumor necrosis factor (TNF)-${\alpha}$; and $58^{\circ}C$ for high mobility group box 1 (HMGB1). Primer efficiencies of all primers calculated for standard curve samples were between 97.1% and 102.6%. No evidence of secondary structure or primer-dimer formation was seen via melt-curve analysis or gel electrophoresis. The developed qRT-PCR assays are highly specific and sensitive and can be used to quantify gene expression levels of canine cytokines.

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