C57BL/6J小鼠脂肪组织总RNA提取及周脂素基因的克隆

Extraction of Total RNA from C57BL/6J Mouse Adipose Tissue and Cloning of Perilipin Gene

目的用改进后的方法提取高质量的RNA,以此为模板,应用T-A克隆法克隆C57BL/6J小鼠周脂素基因编码区,对其进行测序验证,并与GenBank比对。方法在试剂说明书基础上,改进提取脂肪组织RNA的方法,从C57BL/6J附睾脂肪组织提取高质量的总RNA,用RT-PCR扩增出周脂素编码区基因,并将目的基因编码区克隆入pMD18-T载体中,转化E.coli JM109后,筛选阳性克隆,通过限制性内切酶酶切鉴定后,对其进行测序验证,并与GenBank比对。结果用改进后的方法成功提取出了高质量的总RNA,并且成功提取构建的重组载体中含有周脂素基因的全长序列,与GenBank公布的序列一致。结论改进的后的脂肪组织RNA提取方法是可行的,并获得周脂素基因的cDNA,为进一步研究其生物学功能奠定了基础。

Objective Using an improved method to extract high quality total RNA from mouse epididymal adipose tissue,to clone and analyze the full-length cDNA encoding perilipin.Methods According to the specification of reagent,the extraction procedure of total RNA from adipose tissue was modified to extract high quality total RNA from C57BL/6J mouse epididymal adipose tissue.The cDNA encoding perilipin was amplified by RT-PCR using the total RNA.The PCR product was cloned into pMD18-T easy vector and then transformed into E.coli JM109.The recombinant plasmid was identified with restriction enzyme digestion analysis and nucleotide sequencing.Results Using the improved method we successfully extracted high quality total RNA.The recombinant pMD18-T easy vector had a complete open reading frame of perilipin and shared 100% homology with the sequence of mRNA for perilipin reported in GenBank.Conclusions The improved extraction method of total RNA from fat tissue is feasible.The cDNA of perilipin is successfully cloned,which will be helpful for the further research on its biological function.