一种新型的油樟叶片总RNA提取方法

A Novel Method of Total RNA Extraction from Leaves of Cinnamomum longepaniculatum

以油樟叶为试材,比较Trizol法、皂土法、CTAB法、Tris-SDS-异硫氰酸胍法等传统方案提取总RNA,发现效果都不理想。本文针对油樟叶片富含油脂、多酚、多糖的特点,开创性地使用了一种改良的总RNA提取流程:首次增加预处理液、乙酸乙酯抽提来减少油脂、多酚和多糖对裂解液粘稠度的影响,提取过程中使用二氯甲烷代替三氯甲烷增加对疏水性杂质的清除效果,使用混合型高盐溶液多次脱糖,结果表明使用改良的新型方法获得的油樟叶片总RNA条带清晰无明显降解,测得的A260/A280和OD260/OD230均在2.0左右,产物得率约580μg/g,通过RT-PCR扩增出了油樟18S rRNA基因序长为113bp的特异条带,说明这种改良的新型方法获得的RNA完整性、纯度和产率都远高于常规RNA提取方法,研究探索出一种新型油樟叶片总RNA的提取方法。

The leaves of Cinnamomum longepaniculatum are rich in lipids, polyphenols and polysaccharides. Various conventional lysis reagent （Trizol reagents, bentonite, CTAB and Tris-SDS- guanidinium isothiocyanate） methods were tested for extraction of the total Cinnamomum longepaniculatum RNA without ideal results. Based on characters of the leaves containing multiple secondary metabolites to inference RNA purification, a novel modified method was tested： the pretreatment liquid and ethyl acetate were used firstly to reduce lysate fluid viscosity; chloroform was replaced by dichloromethane in RNA extraction process to eliminate effect of hydrophobic impurity; then high concentration of mixed salt were used to take off sugars. Results showed that the new modified method of total RNA extraction from Cinnamomum longepaniculatum leaves was purified, with no obvious degrade, the A260/A280 and A260/A230 were about 2.0, productivity was 580 μg/g- The RNA from the novel method was amplified through RT-PCR and with a 1 13bp specific sequence. The data identified RNA integrity, purity and production efficiency were improved by the novel modified method, and this experiment has determined a new modified method for extraction of total RNA from Cinnamomum longepaniculatum leaves.