适合转录组测序的芒果不同组织RNA试剂盒提取方法研究
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摘要
为了探寻能够满足芒果各组织转录组测序要求的高质量RNA提取方法,以‘桂热芒82号’的叶片、花和果实为材料,从RNA提取质量和产率、琼脂糖电泳和RT-PCR结果分析、RNA测序质量评价这3个方面,对常用的3种试剂盒的提取效果进行了实验研究和评价分析。结果表明:以来自于天根生化科技有限公司的DP 419试剂盒提取的芒果各组织的RNA质量均较好,提取的RNA产率远高于其余两种试剂盒的提取产率;除以美伯生物医药技术有限公司的RM 103试剂盒提取芒果各组织所得RNA,经琼脂糖电泳未见有5S条带出现外,以其余两种试剂盒提取的芒果各组织的RNA都存在28S、18S、5S条带且RT-PCR分析结果理想;对以3种试剂盒提取所得的RNA进行完全测序,结果表明,3种试剂盒提取的RNA完全测序都大于98.99%,能满足RNA-Seq分析的要求。文中综合上述评判因素分析认为,天根生化科技有限公司的DP 419试剂盒是适用于芒果叶片、花和果实RNA提取的最优试剂盒。
In order to study extraction methods of high-quality RNAs from each tissue in Mangifera indica, which could meet the requirement of transcriptome sequencing, leaves, ?owers and fruits of ‘Guiremang No.82' were used as materials, extraction effects of three common kits were researched and evaluated, from three aspects of RNA extraction quality and yield, agarose electrophoresis and RT-PCR results analysis, and RNA sequencing quality evaluation. The results showed that extracted RNAs from all tissues in M. indica had good quality by using the DP419 kit from Tiangen Biochemical Technology Co., Ltd., and extracted RNA yield was much higher than those by using the other two kits.Extracted RNAs from each tissue in M. indica showed no 5 S band through agarose electrophoresis by using the RM103 kit from Meibo Biomedical Technology Co.,Ltd.. Those by using the other two kits had 28 S, 18 S and 5 S bands, and their RT-PCR results were ideal. The results of showed that total RNA sequencing extracted from the three kits was more than98.99%, and could meet the requirement of RNA-seq analysis. In conclusion, the DP419 kit from Tiangen Biochemical Technology Co.,Ltd. was the optimal one for extracting RNAs from leaves, ?owers and fruits in M. indica.
In order to study extraction methods of high-quality RNAs from each tissue in Mangifera indica, which could meet the requirement of transcriptome sequencing, leaves, ?owers and fruits of ‘Guiremang No.82' were used as materials, extraction effects of three common kits were researched and evaluated, from three aspects of RNA extraction quality and yield, agarose electrophoresis and RT-PCR results analysis, and RNA sequencing quality evaluation. The results showed that extracted RNAs from all tissues in M. indica had good quality by using the DP419 kit from Tiangen Biochemical Technology Co., Ltd., and extracted RNA yield was much higher than those by using the other two kits.Extracted RNAs from each tissue in M. indica showed no 5 S band through agarose electrophoresis by using the RM103 kit from Meibo Biomedical Technology Co.,Ltd.. Those by using the other two kits had 28 S, 18 S and 5 S bands, and their RT-PCR results were ideal. The results of showed that total RNA sequencing extracted from the three kits was more than98.99%, and could meet the requirement of RNA-seq analysis. In conclusion, the DP419 kit from Tiangen Biochemical Technology Co.,Ltd. was the optimal one for extracting RNAs from leaves, ?owers and fruits in M. indica.
引文
[1]TONG Z G,QU S C,ZHANG J Y.A modified protocol for RNAextraction from different peach tissues suitable for gene isolation and real-time PCR analysis[J].MolBiotechnol,2012,50:229-236.
[2]林燕,李菲,张淑江,等.适用于转录组测序的大白菜小孢子总RNA提取方法筛选[J].中国蔬菜,2014,1(11):15-19.
[3]井赵斌,魏琳,俞靓,等.转录组测序及其在牧草基因资源发掘中的应用前景[J].草叶科学,2011,28(7):1364-1369.
[4]WANG Z,GERSTEIN M,SNYDER M,RNA-Seq:a revolutionary tool for transcriptomics[J].Nature Research Genetics,2009,10(1):57-63.
[5]刘青柏,刘明国,刘明忠,等.朝阳地区不同品种枣树光合及水分利用特征研究[J].经济林研究,2013,31(3):16-20.
[6]张宇.ISSR分子标记对芒果实生苗亲本鉴别和不同颜色类群遗传多样性分析的研究[D].南宁:广西大学,2009:8.
[7]张育英,陈三阳.热带亚热带果树分类学[M].上海:上海科学技术出版社,1992.
[8]张宇,唐志鹏,高兴,等.不同颜色类群芒果的ISSR分析[J].经济林研究,2014,32(3):72-75.
[9]李桂生.芒果栽培技术[M].广州:广东科技出版社,1993.
[10]张宇,唐志鹏,邓海燕,等.富含多糖多酚芒果果肉组织总RNA的提取[J].湖南农业大学学报(自然科学版),2009,35(6):637-639.
[11]田艳伶,李志辉,杨模华,等.钩栗ISSR-PCR反应体系的建立与优化[J].中南林业科技大学学报,2015,35(2):32-36.
[12]MATKOVICH S J,ZHANG Y,D EREK J,et al.Deep mRNA sequcencing for in vivo functional analysis of card iac transcriptional regulators[J].Circulation Research,2010(6):1459-1467.
[13]张宇,黄国弟,唐志鹏,等.芒果总DNA提取方法比较分析[J].经济林研究,2014,32(2):62-65.
[14]牛美云,程庆伟,张月雅,等.檀香心材总RNA提取方法的比较研究[J].热带亚热带植物学报,2017,25(1):20-25.
[15]荣健,周韬,王平,等.蚬壳花椒种子总RNA提取方法比较与优化[J].中南林业科技大学学报,2017,37(6):60-64.
[16]侯哲,陆秀君,张晓林,等.天女木兰不同组织总RNA提取方法的筛选与优化[J].中南林业科技大学学报,2016,36(7):109-115.
[17]杨峰,易凡,曹慧青,等.长链非编码RNA研究进展[J].遗传,2014,36(5):456-468.
[18]YANG F,YI F,ZHENG Z G,et al.Characterizaton of a carcinogenesis associated long noncoding[J].RNA Biology,2012,9(1):110-116.
[19]MORAN V A,PERERA R J,KHALIL A M.Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs[J].Nucleic Acids Res,2012,40(14):6391-6400.
[20]HEO J B,SUNG S.Vernalization-mediated epigenetic silencing by long intronic noncoding RNA[J].Science,2011,331(6013):76-79.
[2]林燕,李菲,张淑江,等.适用于转录组测序的大白菜小孢子总RNA提取方法筛选[J].中国蔬菜,2014,1(11):15-19.
[3]井赵斌,魏琳,俞靓,等.转录组测序及其在牧草基因资源发掘中的应用前景[J].草叶科学,2011,28(7):1364-1369.
[4]WANG Z,GERSTEIN M,SNYDER M,RNA-Seq:a revolutionary tool for transcriptomics[J].Nature Research Genetics,2009,10(1):57-63.
[5]刘青柏,刘明国,刘明忠,等.朝阳地区不同品种枣树光合及水分利用特征研究[J].经济林研究,2013,31(3):16-20.
[6]张宇.ISSR分子标记对芒果实生苗亲本鉴别和不同颜色类群遗传多样性分析的研究[D].南宁:广西大学,2009:8.
[7]张育英,陈三阳.热带亚热带果树分类学[M].上海:上海科学技术出版社,1992.
[8]张宇,唐志鹏,高兴,等.不同颜色类群芒果的ISSR分析[J].经济林研究,2014,32(3):72-75.
[9]李桂生.芒果栽培技术[M].广州:广东科技出版社,1993.
[10]张宇,唐志鹏,邓海燕,等.富含多糖多酚芒果果肉组织总RNA的提取[J].湖南农业大学学报(自然科学版),2009,35(6):637-639.
[11]田艳伶,李志辉,杨模华,等.钩栗ISSR-PCR反应体系的建立与优化[J].中南林业科技大学学报,2015,35(2):32-36.
[12]MATKOVICH S J,ZHANG Y,D EREK J,et al.Deep mRNA sequcencing for in vivo functional analysis of card iac transcriptional regulators[J].Circulation Research,2010(6):1459-1467.
[13]张宇,黄国弟,唐志鹏,等.芒果总DNA提取方法比较分析[J].经济林研究,2014,32(2):62-65.
[14]牛美云,程庆伟,张月雅,等.檀香心材总RNA提取方法的比较研究[J].热带亚热带植物学报,2017,25(1):20-25.
[15]荣健,周韬,王平,等.蚬壳花椒种子总RNA提取方法比较与优化[J].中南林业科技大学学报,2017,37(6):60-64.
[16]侯哲,陆秀君,张晓林,等.天女木兰不同组织总RNA提取方法的筛选与优化[J].中南林业科技大学学报,2016,36(7):109-115.
[17]杨峰,易凡,曹慧青,等.长链非编码RNA研究进展[J].遗传,2014,36(5):456-468.
[18]YANG F,YI F,ZHENG Z G,et al.Characterizaton of a carcinogenesis associated long noncoding[J].RNA Biology,2012,9(1):110-116.
[19]MORAN V A,PERERA R J,KHALIL A M.Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs[J].Nucleic Acids Res,2012,40(14):6391-6400.
[20]HEO J B,SUNG S.Vernalization-mediated epigenetic silencing by long intronic noncoding RNA[J].Science,2011,331(6013):76-79.