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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。
Using oil camphor leaf as test material, Trizol method, bentonite method, CTAB method and Tris-SDS-guanidinium isothiocyanate method were used to extract total RNA, and the results were not satisfactory. In this paper, aiming at the characteristics of oil-palm leaves rich in oil, polyphenols, polysaccharides, pioneered the use of a modified total RNA extraction process: the first increase of pretreatment solution, ethyl acetate extraction to reduce oil, polyphenols and polysaccharides The effect of the viscosity of the lysate, the use of dichloromethane instead of chloroform to increase the removal of hydrophobic impurities in the extraction process, the use of mixed high-salt solution multiple desolvent, the results show that the use of improved new method of oil camphor The total RNA bands of the leaves were clear and no obvious degradation. The measured A260 / A280 and OD260 / OD230 were about 2.0, the yield of the product was about 580 μg / g. The sequence length of the 18S rRNA gene of C. camphora was 113bp The results showed that the integrity, purity and yield of RNA obtained by this new improved method were much higher than those of conventional RNA extraction methods. A new method of extracting total RNA from camphor leaves was explored.