新生隐球菌总RNA抽提方法的实验研究

目的探讨快速获取高质量的新生隐球菌总RNA的实验方法。方法选取新生隐球菌的荚膜株、荚膜缺陷株,分别设计采用4种方法提取总RNA:酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法。用紫外线分光光度计测量其OD260、OD280的值,并且进行琼脂糖凝胶电泳,同时应用定量PCR法鉴定RNA质量。结果酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法的RNA产量分别为0.2μg/105细胞、0.4μg/105细胞、0.1μg/105细胞、0.6μg/105细胞。结论冷酸洗玻璃珠联合Yeast RNA kit法提取的RNA均一性和完整性最好,是简便、快捷地提取具有荚膜和细胞壁双重屏障的新生隐球菌RNA的理想方法。

Total RNA extraction of Cryptococcus neoformans

Objective A quick approach to extract high quality total RNA from Cryptococcus neoformans plays a vital role in researches on the molecular detection and disease-related gene expression of high pathogenic Cryptococcus neoformans.Method Cryptoccocus neoformans strain and Cryptoccocus neoformans capsule deficient strain were used in the research. Four methods were designed to perform the extraction： acid washed glass beads approach, liquid nitrogen approach, guanidinium isothiocyanate one step approach and acid washed glass beads associated with Yeast RNA kit approach. RNA quality is determined by measurement of OD260 and OD280 with UV spectrophotometer, by agarose gel electrophoresis and by quantitative PCR.Result RNA yield of the four approaches, namely, acid washed glass beads approach, liquid nitrogen approach, guanidinium isothiocyanate one step approach and acid washed glass beads associated with Yeast RNA kit approach, was 0.2 μg/105 cells, 0.4 μg/105 cells, 0.1 μg/105 cells and 0.6 μg/105 cells respectively.Conclusion RNA which gained by the approach of acid washed glass beads associated with Yeast RNA kit has the best homogeneity and integrity. Thus, this method is ideal to perform the extraction of total RNA from Cryptococcus neoformans under protection of capsule and cell wall.