A MODIFIED HOT BORATE METHOD SIGNIFICANTLY ENHANCES THE YIELD OF HIGH-QUALITY RNA FROM COTTON (GOSSYPIUM-HIRSUTUM L)
A MODIFIED HOT BORATE METHOD SIGNIFICANTLY ENHANCES THE YIELD OF HIGH-QUALITY RNA FROM COTTON (GOSSYPIUM-HIRSUTUM L)
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DOI：
10.1006/abio.1994.1538
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发表时间：
1994-11-15
影响因子：
2.9
通讯作者：
WILKINS, TA
中科院分区：
文献类型：
--
作者：
WAN, CY;WILKINS, TA
从棉花中提取具有生物活性的RNA。由于高水平的内源性酚类、多糖和次生代谢物的干扰，很难做到。一种改进的热硼酸法被开发出来，以在组织匀浆过程中对抗这些细胞成分，从而定量回收适合于杂交分析、体外翻译和cDNA合成的RNA。通过吸收光谱、凝胶电泳法、蛋白质合成法和DNA合成法研究了几种热硼酸盐缓冲剂对叶片RNA定性和定量回收的效果。在所评价的缓冲助剂中，聚乙烯吡咯烷酮-40(PVP-40)对棉花叶片RNA的产量和质量的影响是单一的，也是最显著的，尽管加入脱氧胆酸盐和/或脱氧胆酸-40(NP-40)进一步提高了RNA的质量。在碱性pH条件下，用含PVP-40、脱氧胆酸盐和/或NP-40的热硼酸缓冲液从棉花中提取总RNA的定性和定量都是无与伦比的，对其他顽固植物以及特别困难的组织类型也是令人满意的。
The isolation of biologically active RNA from cotton (Gossypium hirsutum L.) is difficult due to interference by high levels of endogenous phenolics, polysaccharides, and secondary metabolites. A modified hot borate procedure was developed to combat these cellular constituents during tissue homogenization, resulting in the quantitative recovery of RNA suitable for hybridization analysis, in vitro translation, and cDNA synthesis. The efficacy of several hot borate buffer adjuvants for the qualitative and quantitative recovery of leaf RNA was monitored by absorbance spectra, gel electrophoresis, protein, and cDNA synthesis. Of the buffer adjuvants evaluated, polyvinylpyrrolidone-40 (PVP-40) exhibited the single, most significant impact on the yield and quality of RNA isolated from cotton leaves, although inclusion of deoxycholate and/or Nonident-40 (NP-40) further enhanced the quality of the RNA. The unsurpassed qualitative and quantitative recovery of total RNA from cotton by hot borate buffer at alkaline pH, supplemented with PVP-40, deoxycholate, and/or NP-40 had also proven satisfactory for other recalcitrant plant species as well as for especially difficult tissue types.