摘要
建立提取完整性好、丰度高的雷公藤RNA的方法，是后续分子生物学试验的必要前提。试验以雷公藤叶、根、愈伤组织和悬浮细胞为材料，比较亚精胺法、SDS法、CTAB法和异硫氰酸胍法从雷公藤组织中提取的RNA，并对最佳方法进行优化。结果表明：SDS法从雷公藤组织中获得RNA片段完整性较好，但丰度较低；对该方法的操作步骤进行删减，并在2次氯仿／异戊醇去除蛋白质和多糖的过程中进行不同时间和程度的振荡提取，得到的RNA较优化前丰度更高，完整性更好，28SrRNA条带的亮度约为18SrRNA条带亮度的2倍，经过紫外分光光度计测定A260／A280比值在1．8～2．0之间，A260／A230比值在1．9～2．1之间。用优化的SDS法提取的雷公藤各组织RNA均具有很好的反转录活性，完全能满足后续RT-PCR、全长基因的克隆和Northernblotting分析等分子生物学研究。
Four methods （Spermidine, sodium dodecyl sulfate, abbreviated as SDS, hexadecyl trimethy am- monium bromide, abbreviated as CTAB, and Guanidinium） were adopted to extract high--quality RNA from the leaves, roots, calli, and suspending cells of Tripterygium wilfordii, Optimalization experiment was conducted. The results demonstrated that the SDA method could extract RNA fragments with high in- tegrity, however, the yield was low. In order to increase the yield, the method was modified. Agarose gel electrophoresis showed that the RNA-containing bands were more clear and bright, and the brightness of 28S rRNA was two times than 18S rRNA. The ratios of D260/D280 and D260/D230 were approximately 1.8-2.0 and 1.9-2.1, respectively. The modified method could be directly used in molecular biology ex- periments.
出处
《西北林学院学报》
CSCD
北大核心
2014年第2期112-116,共5页
Journal of Northwest Forestry University
基金
国家自然科学基金(312710)
关键词
雷公藤
RNA提取
SDS法
方法优化
Tripterygium wilfordii
RNA extraction
SDS method l Optimization
