Establishment of a method for IgM mRNA detecting in green swordtail Xiphophorus helleri by real-time quantitative PCR
ZHANG Ling-ling,REN Yan,SHI Cun-bin,WU Shu-qin
1.School of Life Science and Technology,Dalian Fisheries Univ.,Dalian 116023,China;2.Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China
Abstract: The primers were designed and synthesized in conserved region based on the specific sequence of IgM,and β-actin mRNA.A method to monitor the expression of IgM,and β-actin mRNA was established by real-time quantitative PCR.Total RNA was extracted from the head kidney of green swordtail Xiphophorus helleri immunized with injection,and then cDNA was synthesized by reverse transcription.The target fragments was amplified by PCR and cloned into PMD-18T vector,and the positive recombinant plasmids were used as template to make standard curve and established fluorescence quantitative RT-PCR method.The detection range of Ct value was found to vary from 7 to 21 and from 8 to 25,respectively,and the amplification efficiency to be 2.025 and 1.970,respectively.The melting curve appeared a single peak,with Tm value of 82.43 ℃ and 86.09 ℃,respectively,indicating that the amplification in the quantitative PCR was accurate and specific.In this study,the method established to monitor the expression of gene was used to detect the level of IgM transcription.
2010, Vol. 25 
