【摘要】：目的比較改良SDS法、TRIzol法和CTAB法對湖北釘螺RNA的提取效果,以期獲得一種經(jīng)濟、高效,適于湖北釘螺大樣本RNA的制備方法。方法采用改良SDS法、TRIzol法和CTAB法分別提取湖北釘螺RNA,利用核酸蛋白分析儀測定RNA濃度和純度,通過濃度計算產(chǎn)率,純度以A260/A280、A260/A230表示;以1%瓊脂糖凝膠電泳進一步檢驗RNA完整性;以β-acting為目的基因進行RT-PCR,驗證提取的RNA能否滿足RT-PCR的實驗要求。結(jié)果經(jīng)方差分析,3種方法的產(chǎn)率差異具有統(tǒng)計學(xué)意義(F=16 895.85,P0.01);經(jīng)LSD檢驗,改良SDS法產(chǎn)率較高,其次為TRIzol法,CTAB法產(chǎn)率較低。CTAB法提取的RNA純度較高,A260/A280、A260/A230分別在1.8~2.0和2.0~2.2之間,其余兩種方法的A260/A230均低于2.0。改良SDS法提取的RNA完整性較好,電泳結(jié)果顯示28S、18S和5S條帶完整清晰,條帶之間無明顯彌散現(xiàn)象;而TRIzol法未見28S條帶,CTAB法僅見18S條帶。3種方法提取的RNA經(jīng)RT-PCR均能擴增出陽性條帶。相比另外兩種方法,改良SDS法成本低、耗時少。結(jié)論改良SDS法是一種經(jīng)濟、高效,適于湖北釘螺大樣本RNA的制備方法。
[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.
【作者單位】： 皖南醫(yī)學(xué)院公共衛(wèi)生學(xué)院;
【基金】：安徽省高校優(yōu)秀青年人才支持計劃重點項目(gxyq ZD2016174) 安徽省高校自然科學(xué)研究重點項目(KJ2016A725) 安徽省大學(xué)生創(chuàng)新創(chuàng)業(yè)項目(201510368073、201510368059)
【分類號】：R383.24

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