不同方法提取狗脊蕨叶片总RNA的比较分析

[目的]为构建狗脊蕨叶cDNA文库,保存珍贵基因资源,克隆和研究与有效成分合成相关的基因等提供技术参考。[方法]以狗脊蕨叶片为材料,采用CTAB法、一步法、酚-SDS法和RNA试剂盒提取分离法提取总RNA,通过比较分析确定最优方法。[结果]这4种方法都能提取出182、8 S的RNA,但CTAB法和试剂盒提取分离法的总RNA质量较高,CTAB法还提取出清晰的5S RNA。一步法和SDS法带型模糊,有降解现象。CTAB法提取的狗脊蕨叶片总RNA质量较好,28S1、8S和5S条带清晰,且无明显降解。CTAB提取法OD260/OD280的值在1.93~2.06,试验中其OD260/OD280值为2.012,接近2.0,纯度较高。一步法和SDS法OD260/OD280>2.0,试剂盒法OD260/OD280<2.0。[结论]CTAB法适合提取的狗脊蕨叶片总RNA,质量较好,可用于cDNA合成、文库构建等后续分子生物学试验。

Comparative Analysis of Extracting Total RNA from Woodwardia japonica(L.f.)Sm Leaves by Different Methods

The aim was to provide technical references for constructing cDNA library with high quality of Woodwardia japonica(L.f.)Sm leaf,preserving valuable gene resource,cloning and studying the genes related to available component synthesis.With W.japonica leaf as material,the total RNA was extracted by CTBA method,one step method,phenol-SDS method,extraction and separation method with RNA kit,and the optimum method was confirmed through comparative method.The 18 S RNA and 28 S RNA could be extracted by the 4 methods,but the qualities of total RNA extracted by CTBA method and extraction and separation method with kit were higher,and the clear 5S RNA was extracted by CTBA method.The banding patterns of RNA extracted by one step method and SDS method were fuzzy with some degradation.The quality of total RNA extracted by CTBA method and from W.japonica leaf was better,the bands of 28 S RNA,18 S RNA and 5 S RNA were clear without obvious degradation.The OD260/OD280 value of RNA extracted by CTBA method was at 1.93～2.06 and in the experiment it was about 2.012,near 2.0,showing higher purity.OD260/OD280 values of RNA extracted by one step method and SDS method were bigger than 2.0,and that by kit method was smaller than 2.0.The CTBA method is suitable to extract total RNA of W.japonica leaf,with better quality and can be used in the subsequent molecular biological experiments such as cDNA synthesis and library construction.