A MODIFIED HOT BORATE METHOD SIGNIFICANTLY ENHANCES THE YIELD OF HIGH-QUALITY RNA FROM COTTON (GOSSYPIUM-HIRSUTUM L)

The isolation of biologically active RNA from cotton (Gossypium hirsut um L.) is difficult due to interference by high levels of endogenous p henolics, polysaccharides, and secondary metabolites. A modified hot b erate procedure was developed to combat these cellular constituents du ring tissue homogenization, resulting in the quantitative recovery of RNA suitable for hybridization analysis, in vitro translation, and cDN A synthesis. The efficacy of several hot berate buffer adjuvants for t he 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) ex hibited the single, most significant impact on the yield and quality o f RNA isolated from cotton leaves, although inclusion of deoxycholate and/or Nonident-40 (NP-40) further enhanced the quality of the RNA. Th e unsurpassed qualitative and quantitative recovery of total RNA from cotton by hot berate buffer at alkaline pH, supplemented with PVP-40, deoxycholate, and/or NP-40 had also proven satisfactory for other reca lcitrant plant species as well as for especially difficult tissue type s.