3种方法提取湖北钉螺RNA的比较

发布时间：2018-12-15 20:43

【摘要】：目的比较改良SDS法、TRIzol法和CTAB法对湖北钉螺RNA的提取效果,以期获得一种经济、高效,适于湖北钉螺大样本RNA的制备方法。方法采用改良SDS法、TRIzol法和CTAB法分别提取湖北钉螺RNA,利用核酸蛋白分析仪测定RNA浓度和纯度,通过浓度计算产率,纯度以A260/A280、A260/A230表示;以1%琼脂糖凝胶电泳进一步检验RNA完整性;以β-acting为目的基因进行RT-PCR,验证提取的RNA能否满足RT-PCR的实验要求。结果经方差分析,3种方法的产率差异具有统计学意义(F=16 895.85,P0.01);经LSD检验,改良SDS法产率较高,其次为TRIzol法,CTAB法产率较低。CTAB法提取的RNA纯度较高,A260/A280、A260/A230分别在1.8~2.0和2.0~2.2之间,其余两种方法的A260/A230均低于2.0。改良SDS法提取的RNA完整性较好,电泳结果显示28S、18S和5S条带完整清晰,条带之间无明显弥散现象;而TRIzol法未见28S条带,CTAB法仅见18S条带。3种方法提取的RNA经RT-PCR均能扩增出阳性条带。相比另外两种方法,改良SDS法成本低、耗时少。结论改良SDS法是一种经济、高效,适于湖北钉螺大样本RNA的制备方法。
[Abstract]:Objective to compare the effect of improved SDS method, TRIzol method and CTAB method on the extraction of RNA from Oncomelania hupensis, in order to obtain an economical, efficient and suitable method for the preparation of large sample RNA of Oncomelania hupensis. Methods RNA, of Oncomelania hupensis was extracted by modified SDS method, TRIzol method and CTAB method respectively. The concentration and purity of RNA were determined by nucleic acid protein analyzer. The yield was calculated by the concentration of A260 / A280 A260 / A230. 1% agarose gel electrophoresis was used to further test the integrity of RNA, and RT-PCR, with 尾-acting as the target gene was used to verify whether the extracted RNA could meet the experimental requirements of RT-PCR. Results the results of variance analysis showed that the yield of the three methods had statistical significance (FF16 895.85 P0.01). By LSD test, the yield of modified SDS method was higher than that of TRIzol method, and the yield of CTAB method was lower. The purity of RNA extracted by CTAB method was higher, and A260 / A230 of A260 / A280 / A260 was between 1.80% and 2.0% 2.2, respectively. The A260/A230 of the other two methods is less than 2.0. The integrity of RNA extracted by modified SDS method was good. The results of electrophoresis showed that the bands of 28s 18s and 5s were complete and clear, and there was no obvious dispersion between the bands. However, there were no 28s bands in TRIzol method, but only 18s bands in CTAB method. The positive bands of RNA extracted by three methods could be amplified by RT-PCR. Compared with the other two methods, the modified SDS method is less expensive and time-consuming. Conclusion the improved SDS method is economical, efficient and suitable for the preparation of large sample RNA of Oncomelania hupensis.
【作者单位】：皖南医学院公共卫生学院;
【基金】：安徽省高校优秀青年人才支持计划重点项目(gxyq ZD2016174) 安徽省高校自然科学研究重点项目(KJ2016A725) 安徽省大学生创新创业项目(201510368073、201510368059)
【分类号】：R383.24

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