CAJ | 학술논문

目的探索一种简单有效的人体阴道分泌物标本总RNA提取方法并验证其可行性.方法采用人体上皮细胞与念珠菌标准菌株的混合物(106:106),对匀浆、玻璃珠和液氮研磨三种破壁方法,纯化柱、酚两种提取方法组合产生的5种不同RNA提取方法进行比较.并将提取效果最好的方法应用于临床标本.结果 5种方法均可提取出总RNA,其中,热酚法提取质量浓度最高,达到0.6546 g/L,其次为RNeaay试剂盒提取方法为0.6010 g/L,玻璃珠法为0.2678 g/L,匀浆后TRIzol法为0.2284 g/L,液氮研磨后TRIzol法为0.21 15 g/L.但电泳结果显示,以液氮研磨后TRIzol提取质量最好,而且标准菌株及培养细胞混合物两种细胞成分RNA均被提取,RT-PCR以及荧光定量PCR验证结果显示,该方法可以应用于临床标本RNA提取.结论液氮研磨后TRIzol法是一种简单而有效的阴道分泌物临床标本RNA提取方法,而且提取物可以满足常规下游实验的需求.
목적 탐색일충간단유효적인체음도분비물표본총RNA제취방법병험증기가행성.방법 채용인체상피세포여념주균표준균주적혼합물(106:106),대균장、파리주화액담연마삼충파벽방법,순화주、분량충제취방법조합산생적5충불동RNA제취방법진행비교.병장제취효과최호적방법응용우림상표본.결과 5충방법균가제취출총RNA,기중,열분법제취질량농도최고,체도0.6546 g/L,기차위RNeaay시제합제취방법위0.6010 g/L,파리주법위0.2678 g/L,균장후TRIzol법위0.2284 g/L,액담연마후TRIzol법위0.21 15 g/L.단전영결과현시,이액담연마후TRIzol제취질량최호,이차표준균주급배양세포혼합물량충세포성분RNA균피제취,RT-PCR이급형광정량PCR험증결과현시,해방법가이응용우림상표본RNA제취.결론 액담연마후TRIzol법시일충간단이유효적음도분비물림상표본RNA제취방법,이차제취물가이만족상규하유실험적수구.
Objective To explore a simple and efficient method for RNA extraction from vaginal secretion of patients with vulvovaginal candidiasis and to testify its feasibility.Methods Simulated samples of vaginal secretion were prepared by mixing the same concentration (1×106/L)of human HaCaT keratinocytes and reference Candida spores.RNA was extracted from the simulated sample bv 5 methods,i.e.hot phenol,RNeasy kit,glass beads,grinding in liquid nitrogen+TRzol,homogenate+TRIzol.The quality and quantity of extracted RNA were compared among these methods.The most efficient method was selected and applied to RNA extraction from clinical samples of patients with vulvovaginal candidiasis.Results RNA was extracted successfully from the standard mixture samples by all the 5 methods.The highest mass concentration of total RNA was extracted by hot phenol(0.6546 g/L),followed by RNeasy kit(0.6010 g/L),glass beads (0.2678 g/L),homogenate+TRIzol (0.2284 g/L),gdnding in liquid nitrogen+TRIzol(0.21 15 g/L).Electrophoresis showed that the method of grinding in liquid nitrogen+TRIzol yielded the highest quality of RNA,and it was the only method which could extract RNA from both Candida spores and human cells in this study;moreover,the performance of this method in RNA extraction from clinical samples was veilfled bv RT.PCR and fluorescent quantitative PCR.Conclusions TRIzol after grinding in liquid nitrogen is a simple and effiicient method for RNA extraction from vaginal secretion,with the quality of extracted RNA adequate for following molecular biology research.