Sivakumar S, Franco O L, Thayumanavan B
Centre for Plant Molecular Biology, Tamilnadu Agricultural University, Coimbatore, India.
Prep Biochem Biotechnol. 2007;37(4):323-32. doi: 10.1080/10826060701593217.
Commonly, RNA isolation is the initial step in the study of gene expression analysis and also in the utilization of genes for genetic improvement. However, the recovery of large amounts of RNA with high quality is a difficult process, especially in tissues containing enhanced levels of polysaccharides and other secondary metabolites. Since several procedures for RNA isolation from polysaccharides rich plant tissues have been resulting in poor yields, an effective new protocol is essential for RNA isolation and further analysis. Here, we describe a novel modified technique for isolating total RNA from maturing grains. As a model, we utilized little finger millets, important food staples, which correspond to short duration crops cultivated in varied agro climatic conditions. After isolation, the total RNA was resolved on a denaturing agarose gel, showing more sharp bands of 28S, 18S, and 5S with no degradation. Therefore, the RNA concentration (higher than 1.80) was calculated by spectrophotometry, indicating that RNA is concentrated. Finally, RT-PCR and Northern hybridization confirmed high RNA quality.
通常，RNA提取是基因表达分析研究以及利用基因进行遗传改良的第一步。然而，获得大量高质量的RNA是一个困难的过程，尤其是在含有高水平多糖和其他次生代谢物的组织中。由于从富含多糖的植物组织中提取RNA的几种方法产量都很低，因此一种有效的新方案对于RNA提取和进一步分析至关重要。在这里，我们描述了一种从成熟谷物中分离总RNA的新型改良技术。作为模型，我们使用了小黍，这是重要的主食作物，相当于在不同农业气候条件下种植的短生育期作物。提取后，总RNA在变性琼脂糖凝胶上进行分离，显示出清晰的28S、18S和5S条带，没有降解。因此，通过分光光度法计算出RNA浓度（高于1.80），表明RNA浓度较高。最后，RT-PCR和Northern杂交证实了RNA的高质量。
