一种丹参高质量总RNA的提取方法

A method for high quality of total RNA extracted from Salvia miltorrhiza

高质量RNA的获得是开展丹参分子生物学研究的基础。采用异硫氰酸胍(GuanidineThiocyanate,GT)法、尿素法、CTAB法、苯酚法和热硼酸改良法等五种方法,以丹参组织培养的幼苗为材料,进行丹参RNA的分离试验,发现所采用的几种方法获得的RNA都有不同程度的降解。分析可能是由于丹参中含有大量的多糖以及各种次生代谢成分造成的。在分析现有结果的基础上对GT法进行了改良,在第二次沉淀前附加低浓度乙醇(10%～20%)沉淀20min对于高质量总RNA的获得效果较好。琼脂糖凝胶电泳检测改良后的GT法无论对于组织培养中幼嫩的根、茎、叶还是大田两年生的丹参根、茎、叶和种子均具有很好的RNA分离效果。mRNA电泳检测发现所得mRNA集中分布在500b～3kb之间且质量较高,完全可以满足丹参各种RNA相关的分子生物学实验要求,为丹参RT-PCR、Northern杂交等分子生物学实验提供了良好的基础。

The acquisition of high-quality RNA is a foundation of launching molecule biological study of Salvia miltorrhiza Bunge. But the isolation of RNA from various tissues of Salvia miltorrhiza is notoriously difficult due to the abundance of soluble polyphenolic compounds, amylose and second metabolites. Based on the Guanidine Thiocyanate (GT) method of ZHANG J S, et al, We describe an efficient, practical method for isolation of high quality total RNA pre-cooled guanidine/guanidinium salts. The proteins, genomic DNA and secondary metabolites in the extract were then removed by precipitation with pre-cooled sodium acetate and repeated phenol/chloroform extractions. The RNA was recovered by ethanol precipitation. The RNA obtained from this kind of method is suitable for RT-PCR and northern blot experiments.