Anongpat Suttangkakul, Piyada Juntawong, A. Sirikhachornkit, Chonlada Yaisumlee, Kanidtha Jariyachawalid, K. Kangwansaichol, S. Apisitwanich, S. Vuttipongchaikij
{"title":"An efficient method for isolating large quantity and high quality RNA from oleaginous microalgae for transcriptome sequencing","authors":"Anongpat Suttangkakul, Piyada Juntawong, A. Sirikhachornkit, Chonlada Yaisumlee, Kanidtha Jariyachawalid, K. Kangwansaichol, S. Apisitwanich, S. Vuttipongchaikij","doi":"10.21475/POJ.160902.P7617X","DOIUrl":null,"url":null,"abstract":"Transcriptome analysis requires a large quantity of high-quality DNase-treated RNA for poly(A)+ mRNA isolation and sequencing. This could be problematic in many oleaginous microalgal species that harbor strong cell walls and accumulate high lipid content. Using Scenedesmus obliquus, a microalga with high oil content and potential as a source of algal biofuel, we assessed the efficiency of four RNA isolation methods: direct extraction using TriPure, mechanical breakage using either freeze-thawed with bead beating or grinding in liquid nitrogen followed by TriPure, and grinding in liquid nitrogen before using Qiagen RNeasy Plant Mini Kit. Liquid nitrogen grinding with TriPure method gave the best RNA yields at 15.15 µg mg -1 cell dry weight and ~148.9 µg total RNA from 100 ml culture of S. obliquus. Despite lower yields, RNA isolation of oil accumulating cells (~22% w/w lipid content) provided ~68.1 µg total RNA with the yield of 1.70 µg mg -1 cell dry weight. Transcriptome sequencing and de novo assembly with the average contig length of 824 bp reflected high quality of RNA obtained using this method. The RNA isolation protocol was tested on six other oleaginous microalgae including Chlamydomonas reinhardtii, S. acuminatus, Chorella vulgaris, Chlorococcum humicola, Tetradesmus cumbricus and Coelastrum sp. and yielded 0.86 - 5.42 µg mg -1 cell dry weight. For large scale RNA isolation from microalgae, grinding with liquid nitrogen before TriPure provided the best yield and quality. This finding helps simplify RNA isolation for upcoming transcriptome analyses in microalgae.","PeriodicalId":54602,"journal":{"name":"Plant Omics","volume":"9 1","pages":"126-135"},"PeriodicalIF":0.0000,"publicationDate":"2016-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Omics","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21475/POJ.160902.P7617X","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Agricultural and Biological Sciences","Score":null,"Total":0}
引用次数: 1
Abstract
Transcriptome analysis requires a large quantity of high-quality DNase-treated RNA for poly(A)+ mRNA isolation and sequencing. This could be problematic in many oleaginous microalgal species that harbor strong cell walls and accumulate high lipid content. Using Scenedesmus obliquus, a microalga with high oil content and potential as a source of algal biofuel, we assessed the efficiency of four RNA isolation methods: direct extraction using TriPure, mechanical breakage using either freeze-thawed with bead beating or grinding in liquid nitrogen followed by TriPure, and grinding in liquid nitrogen before using Qiagen RNeasy Plant Mini Kit. Liquid nitrogen grinding with TriPure method gave the best RNA yields at 15.15 µg mg -1 cell dry weight and ~148.9 µg total RNA from 100 ml culture of S. obliquus. Despite lower yields, RNA isolation of oil accumulating cells (~22% w/w lipid content) provided ~68.1 µg total RNA with the yield of 1.70 µg mg -1 cell dry weight. Transcriptome sequencing and de novo assembly with the average contig length of 824 bp reflected high quality of RNA obtained using this method. The RNA isolation protocol was tested on six other oleaginous microalgae including Chlamydomonas reinhardtii, S. acuminatus, Chorella vulgaris, Chlorococcum humicola, Tetradesmus cumbricus and Coelastrum sp. and yielded 0.86 - 5.42 µg mg -1 cell dry weight. For large scale RNA isolation from microalgae, grinding with liquid nitrogen before TriPure provided the best yield and quality. This finding helps simplify RNA isolation for upcoming transcriptome analyses in microalgae.
期刊介绍:
Plant OMICS is an international, peer-reviewed publication that gathers and disseminates fundamental and applied knowledge in almost all area of molecular plant and animal biology, particularly OMICS-es including:
Coverage extends to the most corners of plant and animal biology, including molecular biology, genetics, functional and non-functional molecular breeding and physiology, developmental biology, and new technologies such as vaccines. This journal also covers the combination of many areas of molecular plant and animal biology. Plant Omics is also exteremely interested in molecular aspects of stress biology in plants and animals, including molecular physiology.