Objective: To compare the effects of three kinds of Oncomelania hupensis RNA extraction methods, namely a modified SDS method, TRIzol reagent method, and CTAB method, so as to obtain an economical and efficient method for RNA extraction from O. hupensis.
Methods: The modified SDS method, TRIzol reagent method and CTAB method were applied to extract the RNA from O. hupensis. A nucleic acid protein analyzer was used to measure the concentration and purity of RNA. The yields were calculated by the concentration of the products. The purity was indicated by A260/A280 and A260/A230. The quality of RNA was inspected by 1% agarose gel electrophoresis. The β-acting gene was selected as the target gene for RT-PCR analysis.
Results: The RNA yields obtained by using the three kinds of extraction methods were significantly different (F = 16 895.85, P < 0.01) according to the analysis of variance. The LSD test showed that the yields obtained by using the modified SDS method were the highest, and those obtained by the CTAB method were the lowest. The purity of RNA extracted by the CTAB method was superior to that by the other two methods, and the A260/A280 and A260/A230 ratios of the CTAB method were in the range from 1.8-2.0 and 2.0-2.2. The A260/A230 ratios of the other two methods were both lower than 2.0. The RNA extracted by the modified SDS method had the better integrity. The electrophoresis results showed that the 28S rRNA band, 18S rRNA band and 5S rRNA band were clear, and there was no obvious smear between each band. The RNA obtained by the TRIzol reagent method had no 28S rRNA band, and that obtained by the CTAB method had no 28S rRNA and 5S rRNA bands. The β-acting gene of the RNA extracted by all the three methods could be amplified by RT-PCR. The costs and time-consuming of the modified SDS method were less than those of the other two methods.
Conclusions: The modified SDS method is an economic and efficient method, and it is suitable for extracting the RNA of O. hupensis, especially for large sample preparation.
[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, P < 0.01); 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。.
Keywords: CTAB method; Modified SDS method; Oncomelania hupensis; Quality of RNA; RNA extraction; TRIzol reagent method.