南方红豆杉不同组织总RNA提取方法研究
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摘要
目的:从南方红豆杉不同组织中提取高质量的总RNA。方法:以南方红豆杉幼叶、嫩茎和种子为材料,采用Trizol法、CTAB法、热酚法、改良热酚法和天根RNAprep Pure多糖多酚植物总RNA提取试剂盒法5种方法进行比较分析。结果:Trizol法基本未提取到南方红豆杉不同组织的RNA;CTAB法提取的RNA浓度较高,但OD_(260)/OD_(280)大于2.1,RNA降解严重;热酚法提取的RNA浓度较高,OD_(260)/OD_(280)均小于1.8,蛋白质和酚类物质等残留严重,质量较低;改良热酚法和试剂盒法28S rRNA、18S rRNA和5S rRNA条带清晰,完整性好,OD_(260)/OD_(280)均在1.8~2.1范围内,纯度高。结论:Trizol法、CTAB法和热酚法均不适用于南方红豆杉总RNA提取,而改良热酚法和试剂盒法可提取到质量较好的南方红豆杉总RNA。
Objective: To extract high quality total RNA from different tissues of Taxus mairei(Lemee et LevI.) S. Y. Huex Liu. Methods: The young leaves, tender stems, and seeds of Taxus mairei(Lemee et LevI.) S. Y. Huex Liu were used as materials to extract the total RNA. The five methods, such as Trizol method, CTAB method, hot phenol method, modified hot phenol method and kit method(RNAprep Pure polysaccharide polyphenol plant RNA extraction) were used to compared and analyzed. Results: RNA from different tissues of Taxus mairei(Lemee et LevI.) S. Y. Huex Liu was not extracted by Trizol method. The RNA concentration extracted by CTAB method was high, but OD_(260)/OD_(280) was higher than 2.1, and the RNA degradation was serious. The RNA concentration extracted by hot phenol method was high, and the OD_(260)/OD_(280) was lower than 1.8, but the protein and phenolic substances were seriously residual and of low quality. The bands of 28 s rRNA, 18 s rRNA and 5 s rRNA were clear and had good integrity, which were extracted by modified hot phenol method and kit method, and their OD_(260)/OD_(280) were both within the range of 1.8~2.1 with high purity. Conclusion: The trizol method and CTAB method are not suitable for the extraction of total RNA from Taxus mairei(Lemee et Levl.) S. Y. Huex Liu, while the modified hot phenol method and kit method can extract betterRNA.
Objective: To extract high quality total RNA from different tissues of Taxus mairei(Lemee et LevI.) S. Y. Huex Liu. Methods: The young leaves, tender stems, and seeds of Taxus mairei(Lemee et LevI.) S. Y. Huex Liu were used as materials to extract the total RNA. The five methods, such as Trizol method, CTAB method, hot phenol method, modified hot phenol method and kit method(RNAprep Pure polysaccharide polyphenol plant RNA extraction) were used to compared and analyzed. Results: RNA from different tissues of Taxus mairei(Lemee et LevI.) S. Y. Huex Liu was not extracted by Trizol method. The RNA concentration extracted by CTAB method was high, but OD_(260)/OD_(280) was higher than 2.1, and the RNA degradation was serious. The RNA concentration extracted by hot phenol method was high, and the OD_(260)/OD_(280) was lower than 1.8, but the protein and phenolic substances were seriously residual and of low quality. The bands of 28 s rRNA, 18 s rRNA and 5 s rRNA were clear and had good integrity, which were extracted by modified hot phenol method and kit method, and their OD_(260)/OD_(280) were both within the range of 1.8~2.1 with high purity. Conclusion: The trizol method and CTAB method are not suitable for the extraction of total RNA from Taxus mairei(Lemee et Levl.) S. Y. Huex Liu, while the modified hot phenol method and kit method can extract betterRNA.
引文
[1]中国科学院植物研究所.中国植物志.北京:科学出版社,2005:437-447
[2]张春椿,李影影,黄孝闻,等.南方红豆杉不同部位多糖含量提取分析研究.中华中医药杂志,2014,29(8):2634-2638
[3]李效贤,熊耀康,余陈欢,等.高效液相色谱指纹图谱法分析南方红豆杉药材的氯仿提取物.色谱,2010,28(11):1067-1070
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[5]李石清,张春椿,徐磊,等.响应面分析南方红豆杉枝条多糖超声提取工艺及其抗氧化作用研究.中华中医药杂志,2013,28(10):2942-2946
[6]刘喜君,王仕玉,郭凤根,等.岩白菜幼叶总RNA的提取方法研究.云南农业大学学报(自然科学),2016,30(2):363-367
[7]谢志慧,杜玲玲,李效贤,等.南方红豆杉研究新进展.中国药业,2009,18(15):3-5
[8]Kim J H,Jin H O,Park J A,et al.Comparison of three different kits,for extraction of high-quality RNA from frozen blood.Spring erplus,2014,8(3):76
[9]Tong Z G,Qu S C,Z J Y,et al.Amodified protocol for RNA extraction from different peach tissues suitable for gene isolation and realtime PCR analysis.Mol Biotechnol,2012,50:229-236
[10]Fang G,Hammar S,Grumet R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA.Bio Techniques,1992,13:52-56
[11]Su X,Gibor A.A method for RNA isolation from marinemacro-algae.Anal Biochem,1988,174:650-657
[2]张春椿,李影影,黄孝闻,等.南方红豆杉不同部位多糖含量提取分析研究.中华中医药杂志,2014,29(8):2634-2638
[3]李效贤,熊耀康,余陈欢,等.高效液相色谱指纹图谱法分析南方红豆杉药材的氯仿提取物.色谱,2010,28(11):1067-1070
[4]Wani M C,Taylor H L,Wall M E,et al.Plant antitumor agents.VI.The isolation and structure of taxol,a novel antileukemic and antitumor agent from Taxus brevifolia.J Am Chem Soc,1971,93(9):2325-2327
[5]李石清,张春椿,徐磊,等.响应面分析南方红豆杉枝条多糖超声提取工艺及其抗氧化作用研究.中华中医药杂志,2013,28(10):2942-2946
[6]刘喜君,王仕玉,郭凤根,等.岩白菜幼叶总RNA的提取方法研究.云南农业大学学报(自然科学),2016,30(2):363-367
[7]谢志慧,杜玲玲,李效贤,等.南方红豆杉研究新进展.中国药业,2009,18(15):3-5
[8]Kim J H,Jin H O,Park J A,et al.Comparison of three different kits,for extraction of high-quality RNA from frozen blood.Spring erplus,2014,8(3):76
[9]Tong Z G,Qu S C,Z J Y,et al.Amodified protocol for RNA extraction from different peach tissues suitable for gene isolation and realtime PCR analysis.Mol Biotechnol,2012,50:229-236
[10]Fang G,Hammar S,Grumet R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA.Bio Techniques,1992,13:52-56
[11]Su X,Gibor A.A method for RNA isolation from marinemacro-algae.Anal Biochem,1988,174:650-657