高效提取二相性白假丝酵母菌总RNA实验研究

An Experimental Study on Effective Isolation of Total RNA from Dimorphic Candida albicans

目的:探讨高效、简便提取白假丝酵母菌孢子相及菌丝相细胞总RNA的方法。方法:采用液氮法、超声法及玻璃珠(直径0.55 cm)法提取白假丝酵母菌的孢子相和菌丝相细胞总RNA,紫外分光光度计检测所提取的二相性白假丝酵母菌总RNA的OD260及OD280,琼脂糖凝胶电泳检测所提取的二相性白假丝酵母菌总RNA的完整性。结果:液氮法、超声法及玻璃珠法提取白假丝酵母菌孢子相总RNA的浓度分别为(1.021±0.145)g/L、(0.923±0.031)g/L、(1.121±0.175)g/L,纯度分别为1.697±0.087、1.474±0.064、1.897±0.047;液氮法、超声法及玻璃珠法提取白假丝酵母菌菌丝相总RNA的浓度分别为(0.357±0.260)g/L、(0.287±0.023)g/L、(0.523±0.056)g/L,纯度分别为1.548±0.047、1.474±0.064、1.874±0.064;电泳结果显示,应用玻璃珠法提取的孢子相和菌丝相总RNA 28S rRNA、18S rRNA、5S rRNA三条带均明显显示,而采用液氮法、超声法提取的总RNA三条带不明显。结论:RNAiso Plus试剂联合直径为0.55 cm的玻璃珠法提取的白假丝酵母菌孢子相及菌丝相细胞的总RNA浓度及纯度均较液氮法和超声法好,值得推广。

Objective： To explore an efficient and simple method for extraction of total RNA of Can- dida albicans in yeast form and hyphal form. Methods： The total RNA of Candida albicans in yeast form and hyphal form was isolated by liquid nitrogen method, uhrasonication method and glass bead （diameter 0.55 cm ） method respectively. The OD260 and 00280 values of extracted total RNA were measured by ultraviolet spectrophotometer and agarose gel electrophoresis was carried out to test RNA integrity. Results： The concentrations of yeast form total RNA extracted by liquid nitrogen method, ul- trasonication method and glass bead method were （ 1.021± 0. 145 ） g/L, （0. 923 ± 0.031 ） g/L, （ 1.121 ±0. 175）g/L,and the purities were 1. 697 ±0.087, 1. 474 ±0. 064, 1. 897±0. 047, respectively. The concentrations of hyphal form total RNA extracted by the three methods were （0.357 ±0.260）g/ L, （0. 287 ± 0. 023） g/L, （0. 523 ± 0. 056） g/L, and the purities were 1. 548 ± 0. 047, 1. 474 ± 0. 064, 1. 874 ± 0.064, respectively. The results of electrophoresis showed that there were three clear bands -28S rRNA, 18S rRNA and 5S rRNA when total RNA was isolated with glass bead method and the bands were not clear when RNA was isolated by liquid nitrogen and ultrasonication methods. Con- elusions： The concentration and the purity of total RNA isolated from dimorphic Candida albicans by RNAiso Plus combined with 0.55 cm diameter glass bead method are higher than that isolated by liquid nitrogen and ultrasonication methods. This method is effective, simple, economic and worth populari-zing.