Direct-TRI: High-throughput RNA-extracting Method for all Stages of Zebrafish Development

Abstract

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.