多花蔷薇总RNA提取方法

RNA Isolation from Various Tissues of Rosa multiflora

根据总RNA完整性、纯度和得率筛选出适合多花蔷薇幼嫩根、叶总RNA的提取方法.结果表明,以CTAB/酸酚法提取的扦插苗根系总RNA、以LiCl-尿素法提取的扦插苗根、叶总RNA以及采用RNeasy Plant Mini Kit试剂盒的改进方法提取的组培苗嫩叶总RNA电泳有清晰明亮的28S、18S条带,无降解;其A260/A280值为1.73～2.04,表明总RNA质量好.RT-PCR结果进一步证实所提取的总RNA能够用于分子生物学的各种下游实验.RNA得率分别为:根系和组培苗嫩叶120-140μg/g(fw),扦插苗嫩叶190-230μg/g(fw).CTAB/酸酚法提取的嫩叶总RNA、SDS/酸酚法提取的根、叶总RNA有多糖污染,且有明显降解.Total RNA isolation system(Z5111,Promega)试剂盒不适合提取多花蔷薇各组织总RNA.

The isolation procedure of total RNA from young root and leaf tissue of Rosa multiflora was optimized according to the integrity, purity and yield of RNA. The electrophoresis pattern showed that the quality of RNA isolated from root tissue of cuttage plants with CTAB/acid-phenol protocol and RNA from root and leaf tissue of cuttage plants with LiCl - Urea protocol and RNA from leaf tissue of in vitro cultured plantlets were excellent, shown by clearly visible 28S and 18S ribosomal RNA bands with no apparent degradation and the A260/A280 ratio of the isolated total RNA was 1.73 - 2.04. Isolated RNA was intact and had been proven suitable for further molecular applications by RT-PCR result. These protocols provided us with the yield as 120 ~ 140μg total RNA/g fresh weight of root tissue or in vitro cultured leaf tissue and 190-230μg total RNA/g fresh weight of leaf tissue of cuttage plants）. By contrast, RNA isolated from leaf tissue with CTAB/acid - phenol protocol and RNA isolated from root and leaf tissue with SDS/ acid - phenol protocol contained polysaccharide contamination with apparent degradation. The RNA extraction protocol provided by the kit of total RNA isolation system （ Z5111, Promega） was unsuitable for Rosa multiflora.