阴道念珠菌病患者阴道分泌物总RNA提取方法的研究

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RNeasy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0.2115 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。

Comparison of methods for RNA extraction from vaginal secretion of patients with vulvovaginal candidiasis

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.