Direct-TRI: High-throughput RNA-extracting Method for All Stages of Zebrafish Development
Direct-TRI: High-throughput RNA-extracting Method for All Stages of Zebrafish Development
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DOI：
10.21769/bioprotoc.4136
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发表时间：
2021-09-05
期刊：
影响因子：
0.8
通讯作者：
Hirata, Hiromi
中科院分区：
文献类型：
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作者：
Ujibe, Kota;Nishimura, Kanako;Hirata, Hiromi
最近新一代测序的普及使得转录组分析变得简单。然而，RNA测序之前的大量RNA分离工作以及所涉及的高成本仍然使得大规模转录组分析的常规使用变得困难。例如，传统的苯酚-氯仿 RNA 提取方法无法轻松应用于数百个样品。因此，我们开发了 Direct-TRI，一种新的经济有效的高通量 RNA 提取方法，该方法使用商用胍酚基 RNA 提取试剂和 96 孔硅胶柱板。我们将 Direct-TRI 应用于斑马鱼整个幼虫和幼鱼样本，并通过几种不同的均质方法（例如涡旋、手动均质和冷冻/粉碎）获得了可比较的 RNA 质量。 Direct-TRI 可在一小时内提取 192 个 RNA 样本，每个样本的成本不到 1 美元。 Direct-TRI 可用于大规模转录组研究，可操作数百个斑马鱼个体，并且可用于其他动物样本。
Recent popularization of next-generation sequencing enables conducting easy transcriptome analysis. Nevertheless, substantial RNA isolation work prior to RNA sequencing, as well as the high cost involved, still makes the routine use of large-scale transcriptome analysis difficult. For example, conventional phenol-chloroform RNA extraction cannot be easily applied to hundreds of samples. Therefore, we developed Direct-TRI, a new cost-effective and high throughput RNA-extraction method that uses a commercial guanidine-phenol-based RNA extraction reagent and a 96-well silica column plate. We applied Direct-TRI to zebrafish whole larvae and juvenile samples and obtained comparable RNA qualities by several different homogenization methods such as vortexing, manual homogenizing, and freezing/crushing. Direct-TRI enabled the extraction of 192 RNA samples in an hour with a cost of less than a dollar per sample. Direct-TRI is useful for large-scale transcriptome studies, manipulating hundreds of zebrafish individuals, and may be used with other animal samples.