一种自制的RNA分离试剂及其在鱼组织总RNA提取中的应用

A homemade RNA isolation reagent and its application in the extraction of fish tissue total RNA

【目的】尝试自制一种价格低廉的RNA分离试剂,并检验其应用效果。【方法】以商业化的TRizolReagent为对照,使用自制RNA分离试剂提取草鱼皮肤组织总RNA,采用分光光度计法、变性琼脂糖凝胶电泳法和RT-PCR检测其品质,并用提取的总RNA合成草鱼皮肤cDNA。同时使用自制RNA分离试剂提取斑马鱼总RNA,通过RT-PCR进行E2F5基因全长(1 092bp)的克隆,构建其真核和酵母表达载体并测序。【结果】使用自制RNA分离试剂提取草鱼皮肤组织总RNA的品质与用TRizol Reagent提取的总RNA品质相当,用之成功地合成了草鱼皮肤cDNA。使用自制RNA分离试剂提取得到了更高品质的斑马鱼总RNA,用之成功地克隆了斑马鱼E2F5基因,构建了其真核表达载体pcDNA3.1-flag-Zf E2F5和酵母表达载体pGAD GH-Zf E2F5。【结论】自制RNA分离试剂能够用于鱼类组织总RNA的提取,提取的总RNA能满足后续cDNA合成及RT-PCR克隆特定基因的需要。

【Objective】 The study was done to test a homemade RNA isolation reagent,which is convenient and economical.【Method】 With TRizol Reagent as a comparison,the homemade RNA isolation reagent was used to extract total RNA of grass carp derma.The quality of the total RNA was checked with a spectrophotometer,denaturing agarose gel electrophoresis and also RT-PCR analysis.Finally,the total RNA was used to synthesize grass carp derma cDNA.Subsequently,we applied the homemade reagent to extract total RNA of zebra fish,which was used for RT-PCR to clone the full length zebra fish E2F5 gene（1 092 bp）.Then an eukaryotic expression vector pcDNA3.1-flag-Zf E2F5 and a yeast expression vector pGAD GH-Zf E2F5 were constructed,and the cloned E2F5 gene was sequenced.【Result】 The total RNA of grass carp derma extracted with the homemade RNA isolation reagent was highly qualified as that with TRizol Reagent to synthesize grass carp cDNA.The total RNA of zebra fish extracted with the reagent got a even higher quality,with which the full length E2F5 gene was successfully cloned to construct pcDNA3.1-flag-Zf E2F5 and pGAD GH-Zf E2F5.【Conclusion】 The homemade RNA isolation reagent is suitable for the extraction of fish tissue total RNA,which will satisfy the needs of cDNA synthesis and gene cloning by RT-PCR.With the total RNA extracted using the homemade reagent,we successfully conducted the grass carp cDNA synthesis and zebra fish E2F5 cloning.These will be essential materials for our future studies.