基于Kingfisher Flex系统树木叶片RNA的提取方法比较及优化

Comparison and Optimization of RNA Extraction Methods from Tree Leaves Based on Kingfisher Flex System

随着林木功能基因组学的发展,如何快速高效地提取高质量的核酸变得越来越重要。本研究基于Kingfisher Flex全自动磁珠提取纯化系统,以细叶桉叶片为材料,利用5种磁珠法RNA提取试剂盒(A/B/C/D/E)比较提取RNA的质量和纯度,筛选出试剂盒C为最佳试剂盒。在试剂盒C默认提取程序基础上,利用正交设计L9(34),研究Kingfisher Flex全自动磁珠提取纯化系统上不同洗涤速度、洗脱速度及时间等机器参数对提取RNA的浓度和纯度的影响。正交试验结果表明,对提取RNA的浓度和纯度影响最大的机器参数是洗涤速度,正交试验最优程序组合下提取RNA的纯度稍优于试剂盒C,但是得率仅为试剂盒C的1/3,综合比较后确认试剂盒C默认程序为提取细叶桉叶片RNA的最优提取程序。另外利用试剂盒C也分别对降香黄檀、大果紫檀、油楠、檀香、黑木相思和柚木这六个树种的叶片RNA进行提取,结果表明提取出的RNA均满足cDNA建库以及转录组测序实验的要求。本研究建立了Kingfisher Flex全自动磁珠提取纯化系统上树木叶片RNA的高效提取方法,对树木叶片高通量RNA提取有重要的应用价值,并为其他树种在提取RNA程序上对相关参数进行优化和设置提供了参考。

With the development of tree functional genomics,how to obtain high quality nucleic quickly and efficiently is increasingly important.In this research,based on Kingfisher Flex automatic magnetic bead extraction and purification system,the quality and purity of RNA extracted from Eucalyptus tereticornis Smith leaves were evaluated for five magnetic RNA isolation kits(A/B/C/D/E,with the manufacturers’default programs),and the optimal magnetic RNA isolation kit C was selected.In order to improve the RNA concentration and purity,orthogonal experimental design L9(34)was applied to test the washing speed,elution speed and time on the Kingfisher Flex system.Orthogonal test results indicated that the washing speed was the most important factor affecting the concentration and purity of RNA extracted.Although the purity of RNA extracted with the optimal program of L9(34)was slightly better than the original program of kit C,the RNA yield was only one third of that of its default program.So the default program of kit C was selected as the optimum program for Eucalyptus tereticornis Smith leaf RNA extraction.In addition,the leaves of Dalbergia odorifera T.Chen,Pterocarpus macrocarpus Kurz.,Sindora glabra Merr.,Santalum album L.,Acacia melanoxylon R.Br.and Tectona grandis L.f.were tested for RNA extraction by kit C,and the RNA obtained was suitable for cDNA synthesis and transcriptome sequencing.Thus,the method established here for extracting RNA on Kingfisher Flex system was efficient for tree leaves,which may have important application values for high throughput RNA extraction for tree species.It also provides a useful guideline for RNA extraction with other tree species.