Shukla Ashutosh K, Shasany Ajit K, Khanuja Suman P S
Genetic Resources and Biotechnology Division, Central Institute of Medicinal and Aromatic Plants, Lucknow 226 015, India.
Indian J Exp Biol. 2005 Feb;43(2):197-201.
In the present protocol for extraction of RNA, hexadecyltrimethylammoniumbromide (CTAB) and insoluble polyvinylpyrrolidone were used followed by LiCl precipitation, CsCl ultracentrifugation and finally poly (A)+ mRNA was isolated with the help of oligo(dT)-cellulose columns. The isolated poly (A)+ mRNA was found to be suitable for cDNA-AFLP and suppression subtractive hybridization applications. It is a modified and consolidated protocol based on previously described methods for isolated steps and works better for medicinal and aromatic plants. High yield of poly (A)+ mRNA coupled with its amenability for downstream reactions like RT-PCR, northern blotting and cDNA synthesis for library construction is a key feature of the present protocol.
在目前的RNA提取方案中，使用了十六烷基三甲基溴化铵（CTAB）和不溶性聚乙烯吡咯烷酮，随后进行LiCl沉淀、CsCl超速离心，最后借助寡聚（dT）-纤维素柱分离出聚（A）+ mRNA。发现分离出的聚（A）+ mRNA适用于cDNA-AFLP和抑制性消减杂交应用。这是一个基于先前描述的各分离步骤方法改进和整合的方案，对药用植物和芳香植物效果更好。聚（A）+ mRNA的高产量及其对下游反应（如RT-PCR、Northern印迹和用于文库构建的cDNA合成）的适用性是本方案的一个关键特征。
