柑橘叶片总RNA的两种提取方法比较

Comparison of Two Methods for Extracting Total RNA from Citrus Leaves

为了简便、快速提取高质量的柑橘(Citrus reticulata Banco)叶片总RNA,采用TRlzol法,对北京天根公司RNAplant Reagent和日本TaKaRa公司RNAiso Reagent两种RNA提取试剂进行比较并适当改进。结果表明:通过琼脂糖凝胶电泳,使用TaKaRa公司试剂提取的总RNA28S和18S条带清晰,紫外分光光度计检测分析A260/A280为1.820,A260/A230为2.088,RNA的浓度为2.840μgμl-1,用于RT-PCR反应可成功克隆柑橘β-actin看家基因228 bp片段;采用天根公司试剂,琼脂糖凝胶电泳显示RNA带型较模糊,紫外分光光度计检测分析A260/A280为1.464,A260/A230为1.603,RNA的浓度为2.020μgμl-1,达不到RNA的标准。TaKaRa公司RNAiso Reagent试剂提取的柑橘叶RNA纯度和完整性较好,能用于Northern杂交、cDNA文库的建立及基因克隆等分子生物学实验,为柑橘的进一步分子生物学研究奠定了基础。

The two methods for extracting high quality total RNA from the leaves of Citrus reticulata Banco were compared by using RNAplant Reagent （Beijing Tiangen Corporation） and RNAiso Reagent （Japanese TaKaRa Corporation）, respectively. The results showed that two bands of 28S and 18S rRNA were distincted by agarose gel electrophoresis with RNAiso Reagent, which the values of A260/A280 and A260/A230 were 1. 820 and 2.088, respectively, and the yield reached 2.840 μg ul^-1. At the same time, a citrus house keeping gene β-actin fragment, the 228 bp sequence was successfully cloned by RT-PCR. Otherwise, with RNAplant Reagent, the two RNA bands of 28S and 18S were dim, and the values of A260/A280and A260/A230were 1.464 and 1.603, respectively, the yield of RNA was only 2.020 μg μgμl^-1, which was lower than the standard. Therefore, the RNAiso Reagent was a better method for extracting RNA of Citrus in purity and quality, it could accord with the requirements of subsequent molecular biology reaction, such as Northern hybridization, cDNA libhrary construction and RT-PCR.