适合转录组测序的葡萄叶片总RNA试剂盒提取法的改进

生物技术通报 ›› 2013, Vol. 0 ›› Issue (6): 215-220.

适合转录组测序的葡萄叶片总RNA试剂盒提取法的改进

杨晓燕¹, 张波^{1, 2}, 黄方爱¹, 颜欢¹, 李月荣¹, 郑秋生²

(1.石河子大学药学院, 石河子 832002;2.省部共建新疆特种资源植物药重点实验室, 石河子 832002)

收稿日期:2013-06-20 修回日期:2013-06-20 出版日期:2013-06-20 发布日期:2013-06-20
作者简介:杨晓燕, 女, 硕士研究生, 研究方向: 生物技术制药;E-mail: yeziyxy@sina.com
基金资助:
国家自然科学基金项目(30860032, 31160058), 兵团博士资金项目(2011BB018)

The Improvement on the Protocols of Total RNA Extraction Kits for RNA-Seq from Grape Leaves

Yang Xiaoyan¹ Zhang Bo^{1, 2} Huang Fang'ai¹ Yan Huan¹ Li Yuerong¹ Zheng Qiusheng²

(1. School of Pharmaceutical Sciences, Shihezi University, Shihezi 832002;2. Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education, School of Pharmacy, Shihezi University, Shihezi 832002)

Received:2013-06-20 Revised:2013-06-20 Published:2013-06-20 Online:2013-06-20

摘要/Abstract

摘要： RNA试剂盒法是获取植物高质量RNA样品的常用技术, 针对3种常见RNA提取试剂盒在红地球葡萄叶样品RNA提取效果进行评估和优化, 以期找到适合红地球葡萄叶片RNA表达谱测序需求的高质量RNA提取策略。试验依据3种试剂盒推荐时间和条件进行RNA提取, 并对时间等条件进行优化, 比对了优化前后RNA质量。结果显示, 试剂盒经优化后的方法提取的RNA收率高, 完整性好, OD₂₆₀/OD₂₈₀在1.8-2.2之间, OD₂₆₀/OD₂₃₀大于2, 28S与18S条带清晰完整, RNA-Seq测序质量及基因覆盖度符合要求。在红地球葡萄叶片上的RNA提取结果表明, 延长提取试剂与样品的接触时间操作可以显著提高RNA提取质量(纯度/完整性) 。

关键词: 红地球葡萄, RNA提取, 试剂盒法, 转录组短枪测序法

Abstract: Commercial RNA extraction kits were wildly used in obtaining high quality RNA samples from plants. To seek the optimal method for the extraction of high quality RNA in RNA Expression Sequences Array(RNA-Seq), the topic of present article is to evaluate and optimize the effects of three common RNA extraction kits on leaves samples from grapevine(Vitis vinifera cv. Red Globe). The quantities of RNA were analyzed between the recommendatory and optimal usages of three kits in term of times. Results showed that the optimized procedure for RNA extraction displayed higher yield and better integrity on total RNA. There are a ratios of OD₂₆₀/OD₂₈₀ from 1.8 to 2.2 and further OD₂₆₀/OD₂₃₀ratio no less than 2, resulted in a integrate 28S and 18S bands by electrophoresis. The extracted samples also kept their quality and gene coverage from RNA-Seq sequencing. Our research demonstrated that the prolonged incubation time for samples preparation and other procedures would significantly benefit the separation of total RNA from grape leaves(Vitis vinifera cv. Red Globe)with a higher purity and integrity.

Key words: Vitis vinifera cv., Red Globe, RNA extaction, Kit assay, RNA-Seq

杨晓燕, 张波, 黄方爱, 颜欢, 李月荣, 郑秋生. 适合转录组测序的葡萄叶片总RNA试剂盒提取法的改进[J]. 生物技术通报, 2013, 0(6): 215-220.

Yang Xiaoyan, Zhang Bo, Huang Fang'ai, Yan Huan, Li Yuerong, Zheng Qiusheng. The Improvement on the Protocols of Total RNA Extraction Kits for RNA-Seq from Grape Leaves[J]. Biotechnology Bulletin, 2013, 0(6): 215-220.

参考文献

[1] Tong ZG, Qu SC, Zhang JY. A Modified protocol for RNA extraction from different peach tissues suitable for gene isolation and real-time PCR analysis [J]. Mol Biotechnol, 2012, 50:229-236.
[2] 张卉, 王文全, 杨全. 甘草RNA提取方法研究 [J]. 时珍国医国药, 2007, 12(18):3051-3052.
[3] 张宇玉, 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, 2009, 6(35):637-639.
[4] 郭翠英, 王跃进, 刘雅丽, 等. 富含多糖葡萄风信子花瓣总RNA 提取方法研究 [J]. 西北农业学报, 2007, 16(3):188-191.
[5] 杨继涛, 张素勤, 耿广东, 等. 辣椒叶片总RNA提取与质量分析 [J]. 西北农业学报, 2009, 18(6):217-220.
[6] Hemanth KNV, Ramesh K, Mehboob BS. Efficient protocol for isolation of functional RNA from different grape tissue rich in polyphenols and polysaccharides for gene expression studies [J]. Electronic Journal of Biotechnology, 2008, 3(11):1-8.
[7] 张今今, 王跃进, 王西平, 等. 葡萄总RNA提取方法的研究 [J]. 果树学报, 2003, 20(3):178-181.
[8] Iandolino AB, GOES da SILVA F, Lim H, et al. High-quality RNA, cDNA, and derived EST libraries from grapevine(Vitis vinifera L.)[J]. Plant Molecular Biology Reporter, 2004, 22:269-278.
[9] 房经贵, 高志红, 陶建敏. 一种提取葡萄芽中总RNA 的方法 [J]. 生物技术, 2003(2):24-25.
[10] 李红熙, 徐美隆, 杨智. 一种适合葡萄多种组织的总RNA提取方法 [J]. 中国农学通报, 2012, 28(7):155-159.
[11] Matkovich SJ, Zhang Y, Derek J, et al. Deep mRNA sequencing for in vivo functional analysis of cardiac transcriptional regulators [J]. Circulation Research, 2010(6):1459-1467.
[12] Loulakakis KA, Roubelakis-Angelakis KA, Kanellis AK. Isolation of functional RNA from grapevine tissues poor in nucleic acid content [J]. American Journal Enology and Viticulture, 1996, 47:181-185.
[13] Schultz DJ, Craig R, Cox-Foster DL, et al. RNA isolation from recalcitrant plant tissue [J]. Plant Molecular Biology Reporter, 1994, 12(4):310-316.
[14] Lorenz WW, Yu YS, Dean JFD, et al. An improved method of RNA isolation from loblolly pine(P. taeda L.)and other conifer species [J]. Journal of Visualized Experiments, 2010, 22(36):1751-1756.
[15] Zenoni S, Ferrarini A, Giacomelli E, et al. Characterization of transcriptional complexity during berry development in Vitis vinifera using RNA-Seq [J].Plant Physiology, 2010, 152(4):1787-1795.
[16] Severin A J, Woody JL, Bolon YT, et al. RNA-Seq Atlas of Glycine max:A guide to the soybean transcriptome [J].Plant Biology, 2010, 10:160-176