牡丹种子总RNA提取方法比较
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摘要
采用CTAB法、Trizol法及改进的RNA提取试剂盒方法提取牡丹种子的总RNA,比较三种方法的优劣,从而筛选出一种适合于牡丹种子高质量RNA的提取方法。结果表明,以RNA提取试剂盒为基础,当每管样品是25 mg(700μL裂解液)时,六管合一管过吸附柱的方法操作简单,且能够有效地去除牡丹种子中脂肪、多糖、多酚等代谢物质,从而提取出高质量的RNA。琼脂糖凝胶电泳检测结果显示该法提取的RNA完整性好,紫外分光光度计检测其OD260/OD280比值为2.03。而且,该法提取得到的RNA被成功应用于荧光定量PCR表达分析等后续的分子生物学研究。
Total RN A from Paeonia suffruticosa seeds was extracted by Trizol method, CTAB method and improved RNA prep Pure Plant Kit method, respectively. The three different methods were compared and the most effective method of isolation of high-quality Paeonia suffruticosa RNA was determined. The results showed that on the basis of RNA Kit method, when the amount of sample was 25 mg per tube(700 μL Lysis Buffer) and all RNA lysate in six tubes were collected into one column, the improved kit method could get high-quality RNA from seeds, which was simple and effective to remove the metabolites such as fat, polysaccharides, polyphenols. The RNA isolated with this method showed good integrity by agarose gel electrophoresis detection and gave an OD260/OD280 ratio of 2.03 by spectrophotometric detection. Furthermore, the RNA extracted with this method was proved to be successfully applied for fluorescent quantitative PCR analysis and other downstream applications in molecular biology research.
Total RN A from Paeonia suffruticosa seeds was extracted by Trizol method, CTAB method and improved RNA prep Pure Plant Kit method, respectively. The three different methods were compared and the most effective method of isolation of high-quality Paeonia suffruticosa RNA was determined. The results showed that on the basis of RNA Kit method, when the amount of sample was 25 mg per tube(700 μL Lysis Buffer) and all RNA lysate in six tubes were collected into one column, the improved kit method could get high-quality RNA from seeds, which was simple and effective to remove the metabolites such as fat, polysaccharides, polyphenols. The RNA isolated with this method showed good integrity by agarose gel electrophoresis detection and gave an OD260/OD280 ratio of 2.03 by spectrophotometric detection. Furthermore, the RNA extracted with this method was proved to be successfully applied for fluorescent quantitative PCR analysis and other downstream applications in molecular biology research.
引文
Din g Y.,Liu Y.,Yang Y.Y.,and Gan L.,2006,An efficien method for extraction of high quality RNA from Rapeseeds Huazhong Nongye Daxue Xuebao(Journal of Huazhong Agricultural University),25(5):465-468(丁勇,刘英,杨鸯鸯甘莉,2006,油菜种子高质量总RNA提取的一种有效方法,华中农业大学学报,25(5):465-468)
Hu C.Q.,Gao J.Y.,and Chen H.B.,2007,A simple method fo peanut seeds total RNA extraction,Shipin Kexue(Food Sci ences),28(10):373-375(胡纯秋,高金燕,陈红兵,2007,一种提取花生种子中总RNA的简易方法,食品科学,28(10):373-375)
Hu J.,Tan X.F.,Zhang L.,and Long H.X.,2010,Extraction o total RNA and identification of Cod8FAD gene in Camelli oleifera seeds,Zhejiang Linye Keji(Journal of Zhejiang Fo Sciences and Technology),30(2):17-21(胡姣,谭晓风,张琳,龙洪旭,2010,油茶种子总RNA提取及Cod8FAD基因的鉴定,浙江林业科技,30(2):17-21)
Jiang S.H.,Li Y.H.,Wang J.Q.,Liang F.,Wang M.F.,and Cui B.2015,Comparison research of extraction method of tota RNA from Sedirea japonica,Henan Nongye Daxue Xuebao(Journal of Henan Agricultural University),49(4):468-471(蒋素华,李艳辉,王洁琼,梁芳,王默霏,崔波,2015,萼脊兰总RNA提取方法的比较研究,河南农业大学学报,49(4):468-471)
Li H.,and Wang X.L.,1999,The difficulties in the isolation o RNA from plant tissues and their resolving strategies,Sheng wu Jishu Tongbao(Biotechnology Information),(1):36-39(李宏,王新力,1999,植物组织RNA提取的难点及对策生物技术通报,(1):36-39)
Liu J.Y.,ed.,2002,Molecular biology experiment instruction Tsinghua University Press,Beijing,China,pp.59-61(刘进元,编著,2002,分子生物学实验指导,清华大学出版社中国,北京,pp.59-61)
Su X.,and Gibor A.,1988,A method for RNA isolation from marine macro-algae,Anal.Biochem.,174(2):650-657
Sun H.B.,Wang Z.H.,Liu X.Q.,Huang J.Y.,Dong C.H.,and Liu S.Y.,2012,Modified RNA extraction method for seeds o rapeseed,soybean,peanut and sesame,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),34(4):353-358(孙海波,王智慧,刘学群,黄军艳,董彩华,刘胜毅,2012,一种适用于油菜、大豆、花生、芝麻四种油料作物种子的RNA提取方法,中国油料作物学报,34(4)353-358)
Xu B.B.,Xie L.L.,Yan H.,He Y.,and Tian Z.H.,2007,Research on the methods of isolation of RNA from oilseeds(Brassic napus.L),Heilongjiang Nongye Kexue(Heilongjiang Agri cultural Sciences),(5):18-20(许本波,谢伶俐,严寒,何勇田志宏,2007,甘蓝型油菜籽粒RNA提取方法的探讨,黑龙江农业科学,(5):18-20)
Yuan M.Z.,Wen R.,Liu J.S.,Jie Y.S.,Li J.H.,Yin H.P.,Zeng QL.,Long G.,Yang D.H.,Liu H.,Huang S.Q.,and Xu J.2005,Isolation of total RNA from different plant species Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),3(2)285-292(袁明珠,温柔,刘吉升,掲英省,黎金花,尹汗萍曾秋莲,龙刚,杨德华,刘海,黄胜琴,徐杰,2005,几种植物材料中总RNA的提取,分子植物育种,3(2):285-292)
Zhang T.,Gao T.S.,Bai R.Y.,Huang X.L.,Guo N.,Lu J.X.,and Luo A.C.,2015,Utilization and research progress of oil tre peony,Chongqing Shifan Daxue Xuebao(Journal of Chong qing Normal University(N atural Science)),32(2):143-149(张涛,高天姝,白瑞英,黄兴琳,郭楠,陆俊杏,罗安才2015,油用牡丹利用与研究进展,重庆师范大学学报(自然科学版),32(2):143-149)
Zhang Y.L.,Han X.Y.,Niu L.X.,Zhang J.,and He L.X.,2015Analysis of fatty acid in seed oil from nine wild peony species,Zhongguo Liangyou Xuebao(Journal of the Chi nese Cereals and Oils Association),30(4):72-79(张延龙,韩雪源,牛立新,张静,何丽霞,2015,9种野生牡丹籽油主要脂肪酸成分分析,中国粮油学报,30(4):72-79)
Zhao C.X.,Ma L.H.,and Yang T.W.,2008,Extraction of tota RNA in Aloe vera L.var.chinensis leaf with improved CTAB method,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),36(16):6671-6672(赵春喜,马利华杨同文,2008,改进的CTAB法提取中华芦荟叶总RNA安徽农业科学,36(16):6671-6672)
Zhou H.M.,Ma J.Q.,Miao C.Y.,Hu J.L.,Yang Z.Y.,Li P.,and Zheng M.L.,2009,Physicochemical indexes and fatty acid composition of peony seed oil,Zhongguo Youzhi(Chin Oils and Fats),34(7):72-74(周海梅,马锦琦,苗春雨,胡吉良,杨志勇,李朴,郑美玲,2009,牡丹籽油的理化指标和脂肪酸成分分析,中国油脂,34(7):72-74)
Hu C.Q.,Gao J.Y.,and Chen H.B.,2007,A simple method fo peanut seeds total RNA extraction,Shipin Kexue(Food Sci ences),28(10):373-375(胡纯秋,高金燕,陈红兵,2007,一种提取花生种子中总RNA的简易方法,食品科学,28(10):373-375)
Hu J.,Tan X.F.,Zhang L.,and Long H.X.,2010,Extraction o total RNA and identification of Cod8FAD gene in Camelli oleifera seeds,Zhejiang Linye Keji(Journal of Zhejiang Fo Sciences and Technology),30(2):17-21(胡姣,谭晓风,张琳,龙洪旭,2010,油茶种子总RNA提取及Cod8FAD基因的鉴定,浙江林业科技,30(2):17-21)
Jiang S.H.,Li Y.H.,Wang J.Q.,Liang F.,Wang M.F.,and Cui B.2015,Comparison research of extraction method of tota RNA from Sedirea japonica,Henan Nongye Daxue Xuebao(Journal of Henan Agricultural University),49(4):468-471(蒋素华,李艳辉,王洁琼,梁芳,王默霏,崔波,2015,萼脊兰总RNA提取方法的比较研究,河南农业大学学报,49(4):468-471)
Li H.,and Wang X.L.,1999,The difficulties in the isolation o RNA from plant tissues and their resolving strategies,Sheng wu Jishu Tongbao(Biotechnology Information),(1):36-39(李宏,王新力,1999,植物组织RNA提取的难点及对策生物技术通报,(1):36-39)
Liu J.Y.,ed.,2002,Molecular biology experiment instruction Tsinghua University Press,Beijing,China,pp.59-61(刘进元,编著,2002,分子生物学实验指导,清华大学出版社中国,北京,pp.59-61)
Su X.,and Gibor A.,1988,A method for RNA isolation from marine macro-algae,Anal.Biochem.,174(2):650-657
Sun H.B.,Wang Z.H.,Liu X.Q.,Huang J.Y.,Dong C.H.,and Liu S.Y.,2012,Modified RNA extraction method for seeds o rapeseed,soybean,peanut and sesame,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),34(4):353-358(孙海波,王智慧,刘学群,黄军艳,董彩华,刘胜毅,2012,一种适用于油菜、大豆、花生、芝麻四种油料作物种子的RNA提取方法,中国油料作物学报,34(4)353-358)
Xu B.B.,Xie L.L.,Yan H.,He Y.,and Tian Z.H.,2007,Research on the methods of isolation of RNA from oilseeds(Brassic napus.L),Heilongjiang Nongye Kexue(Heilongjiang Agri cultural Sciences),(5):18-20(许本波,谢伶俐,严寒,何勇田志宏,2007,甘蓝型油菜籽粒RNA提取方法的探讨,黑龙江农业科学,(5):18-20)
Yuan M.Z.,Wen R.,Liu J.S.,Jie Y.S.,Li J.H.,Yin H.P.,Zeng QL.,Long G.,Yang D.H.,Liu H.,Huang S.Q.,and Xu J.2005,Isolation of total RNA from different plant species Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),3(2)285-292(袁明珠,温柔,刘吉升,掲英省,黎金花,尹汗萍曾秋莲,龙刚,杨德华,刘海,黄胜琴,徐杰,2005,几种植物材料中总RNA的提取,分子植物育种,3(2):285-292)
Zhang T.,Gao T.S.,Bai R.Y.,Huang X.L.,Guo N.,Lu J.X.,and Luo A.C.,2015,Utilization and research progress of oil tre peony,Chongqing Shifan Daxue Xuebao(Journal of Chong qing Normal University(N atural Science)),32(2):143-149(张涛,高天姝,白瑞英,黄兴琳,郭楠,陆俊杏,罗安才2015,油用牡丹利用与研究进展,重庆师范大学学报(自然科学版),32(2):143-149)
Zhang Y.L.,Han X.Y.,Niu L.X.,Zhang J.,and He L.X.,2015Analysis of fatty acid in seed oil from nine wild peony species,Zhongguo Liangyou Xuebao(Journal of the Chi nese Cereals and Oils Association),30(4):72-79(张延龙,韩雪源,牛立新,张静,何丽霞,2015,9种野生牡丹籽油主要脂肪酸成分分析,中国粮油学报,30(4):72-79)
Zhao C.X.,Ma L.H.,and Yang T.W.,2008,Extraction of tota RNA in Aloe vera L.var.chinensis leaf with improved CTAB method,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),36(16):6671-6672(赵春喜,马利华杨同文,2008,改进的CTAB法提取中华芦荟叶总RNA安徽农业科学,36(16):6671-6672)
Zhou H.M.,Ma J.Q.,Miao C.Y.,Hu J.L.,Yang Z.Y.,Li P.,and Zheng M.L.,2009,Physicochemical indexes and fatty acid composition of peony seed oil,Zhongguo Youzhi(Chin Oils and Fats),34(7):72-74(周海梅,马锦琦,苗春雨,胡吉良,杨志勇,李朴,郑美玲,2009,牡丹籽油的理化指标和脂肪酸成分分析,中国油脂,34(7):72-74)