铁皮石斛花总RNA提取方法的比较研究

Comparison of Several Total RNA Extraction Methods from Flowers of Dendrobium officinale

摘要:

为筛选铁皮石斛（Dendrobium officinale）花总RNA提取方法，对8种提取方法进行了比较研究，包括改良CTAB-LiCl法（M1）、改良CTAB-异丙醇法（M2）、改良SDS-LiCl法（M3）、改良SDS-异丙醇法（M4）、多糖多酚植物RNA提取试剂盒法（M5）、柱式植物RNAout 2.0试剂盒法（M6）、RNAprep Pure多糖多酚植物总RNA提取试剂盒法（M7）和Biospin多糖多酚植物总RNA提取试剂盒法（M8）。结果表明，以M4和M5提取的总RNA带型清晰，完整性好，A_{260 nm}/A_{280 nm}为1.8~2.0，A_{260 nm}/A_{230 nm}大于2.0，RNA产率分别为（159.45±1.45）和（170.84±3.53）μg/g。利用M4、M5提取霍山石斛、金钗石斛、鼓槌石斛和美花石斛花的总RNA，样品的完整性、浓度和纯度均符合质量要求。以M4、M5提取的铁皮石斛总RNA为模板，扩增Actin基因片段，扩增产物大小与预期一致且条带单一。这说明M4、M5方法操作简便，结果重复性好，能够较好地提取石斛属植物花的总RNA。

Abstract:

In order to screen total RNA extraction method from flowers of Dendrobium officinale, eight methods were compared, including improved CTAB-LiCl method (M1), improved CTAB-isopropanol method (M2), improved SDS-LiCl method (M3), improved SDS-isopropanol method (M4), Quick RNA Isolation Kit (Huayueyang, China; M5), Column Plant RNAout 2.0 Kit (Tiandz, China; M6), RNAprep Pure Plant Plus Kit (Tiangen, China; M7) and Plant Total RNA Extraction Kit (Biospin, China; M8). The results showed that the integrity of total RNA extracted using M4 and M5 was good, with distinct electrophoresis bands of 28S and 18S rRNA. The spectrophotometric values of A_{260 nm}/A_{280 nm} ranged from 1.8 to 2.0, and the A_{260 nm}/A_{230 nm} value was more than 2.0. The yields of total RNA extracted using M4 and M5 were (159.45±1.45) and (170.84±3.53) μg/g, respectively. Additionally, the total RNA of D. huoshanense, D. nobile, D. chrysotoxum and D. loddigesii extracted by M4 and M5 was verified to meet the quality requirements on integrity, purity and concentration. The total RNA of D. officinale extracted by M4 and M5 was used as template, and the amplified Actin gene showed a single band, consisting with the prediction. Therefore, M4 and M5 were effective methods to extract total RNA from flowers of Dendrobium plants, especially D. officinale.