An efficient method for isolating large quantity and high quality RNA from oleaginous microalgae for transcriptome sequencing

Q3 Agricultural and Biological Sciences
Anongpat Suttangkakul, Piyada Juntawong, A. Sirikhachornkit, Chonlada Yaisumlee, Kanidtha Jariyachawalid, K. Kangwansaichol, S. Apisitwanich, S. Vuttipongchaikij
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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.
从产油微藻中分离大量高质量RNA进行转录组测序的有效方法
转录组分析需要大量高质量的dna处理RNA进行poly(a)+ mRNA的分离和测序。这在许多富含油脂的微藻物种中可能是有问题的,这些微藻拥有坚固的细胞壁并积累了高脂质含量。我们利用一种含油量高且有潜力作为藻类生物燃料来源的微藻Scenedesmus obliquus,评估了四种RNA分离方法的效率:使用TriPure直接提取,使用冷冻解冻和打珠或在液氮中研磨后再使用TriPure,以及在使用Qiagen RNeasy Plant Mini Kit之前先在液氮中研磨。用TriPure方法进行液氮研磨的结果表明,100 ml培养的斜棘鱼的RNA产量为15.15µg mg -1细胞干重,总RNA产量为~148.9µg。尽管产率较低,但产油细胞(~22% w/w脂含量)的RNA分离提供了~68.1µg总RNA,产率为1.70µg mg -1细胞干重。转录组测序和从头组装平均contig长度为824 bp,反映了使用该方法获得的高质量RNA。该RNA分离方案在reinhardtii衣藻、S. acuminatus、Chorella vulgaris、chlorcoccum humicola、Tetradesmus cumbricus和Coelastrum sp.等6种产油微藻上进行了测试,细胞干重为0.86 ~ 5.42µg mg -1。对于从微藻中大规模分离RNA,在TriPure之前用液氮研磨可以提供最好的产量和质量。这一发现有助于简化即将在微藻中进行转录组分析的RNA分离。
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来源期刊
Plant Omics
Plant Omics 生物-植物科学
CiteScore
1.30
自引率
0.00%
发文量
0
审稿时长
6 months
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.
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