适合于高通量测序的槟榔总RNA的提取方法

Efficient Isolation of Total RNA from Areca Leaf for High-throughput Sequencing

为了获得能满足高通量测序要求的槟榔叶片总RNA,采用通用植物总RNA提取试剂盒和异硫氰酸胍-苯酚法提取槟榔叶片的总RNA。结果表明:异硫氰酸胍-苯酚法提取的总RNA完整性好、纯度高,通用植物总RNA提取试剂盒不能从槟榔叶片中提取完整的总RNA;70℃,10 min的热处理会导致槟榔总RNA的降解,苯酚/氯仿抽提能得到高纯度、完整性好的总RNA;总RNA质量浓度达到979 mg·L-1,OD260/OD280=1.98,OD260/OD230=2.31,能满足转录组高通量测序的要求。

Total RNA of leaves of Areca nut （Areca catechu L） were extracted by using universal plant total RNA extraction kit and the guanidine isothiocyanate-phenol method to develop an optimum total RNA extraction protocol for Areca leaves for high-throughput sequencing. A better quality of Areca leaf total RNA was extracted by the guanidine isothiocyanate- phenol than the universal plant total RNA extraction kit. Furthermore, two methods, heat treatment and phenol-chloroform extraction, were tried to inactivate DNAase I after total RNA was digested by this enzyme. It was found that the total leaf RNA was degraded when heat treated at 70℃ for 10min, whereas high-purity and high concentration of total leaf RNA was obtained through phenol-chloroform extraction. The quality of total RNA with 0D26o/0D280 = 1.98, 0D260/OD230 = 2.31 and concentration more than 979 mg·L-1, met the standard requirements for high-throughput RNA sequencing.