从少量培养细胞中同时提取微量蛋白和RNA的方法探讨

为建立一项从少量培养细胞中同时提取RNA和蛋白质的技术 ,向 2～ 3× 10 5细胞中加入 1ml自制RNA提取试剂 ,RNA抽提后剩下的中下两相 ,用异丙醇、盐酸胍和无水乙醇抽提蛋白质 .同时用进口Tripure试剂、经典的异硫氰酸胍 苯酚 氯仿一步抽提RNA法和分子克隆实验手册裂解液制备蛋白质的方法 ,作为对照 .自制试剂提取的总RNA ,18S、2 8S清晰可见 ,2 8S比 18S带亮度强 2～ 3倍 ,带与带之间无拖尾现象 ,5S隐约可见 ,而且成功地进行了Northern印迹、RT PCR分析 ,与经典方法差异不大 ;用此法所提蛋白质 ,经SDS PAGE检测 ,蛋白分离效果很好 ,无杂质 ,且Western印迹检测Giα蛋白 ,可见一条清晰的特异带 ,与常规提取蛋白质 ,结果相似 .从微量细胞中同时提取的RNA和蛋白质 ,得率高、纯度好 ,具有化学完整性和生物学性质

Extraction of Trace Total RNA and Protein from Small Quantity Cultured Cells

A technique for extracting trace total RNA and protein from a small quantity of cultured cells was developed.1 ml homemade RNA extraction reagent was added to the culture dish containing about 2～3×10+5 cells/well. Then, the cell lysate was transferred to a polypropylene centrifuge tube,incubated for 5 min at room temperature,extracted with chloroform.RNA was deposited with isopropanol.From the phenol-ethanol supernatant,protein was isolated by adding isopropanol,0.3 mol/L guanidine hydrochloride in 95% ethanol,and 100% ethanol.The total RNA isolated with homemade extraction reagent was not degraded and free of protein and DNA.The isolated total RNA had an A 260/A 280 ratio of 1.8～2.0.It was successfully analyzed by Northern blotting and RT-PCR.The isolated protein was separated by SDS-PAGE with high purity.It had an A 280/A 260 ratio of 1.8～2.0 and was successfully analyzed by Western blotting.The extraction method provides high yield and purity for the total RNA and protein isolated from a small quantity of cultured cells,which are suitable for Northern blot analysis,RT-PCR analysis and Western blot analysis.