Background

RNA-sequencing (RNA-Seq) is one of the standard methods in various fields of biology (Cheng et al., 2021; Sun et al., 2021) because it provides comprehensive information about gene expression. Recently, several researchers have developed cost-effective and high-throughput RNA-Seq library preparation protocols such as Lasy-Seq (Kamitani et al., 2019), BRB-Seq (Alpern et al., 2019), and Decode-Seq (Li et al., 2020). Although these techniques and the lower cost of massive parallel sequencing have enabled large-scale RNA-Seq analyses (Hoang et al., 2020; Miller et al., 2013), the RNA extraction process prior to RNA-Seq remains laborious and time-consuming. For example, a conventional RNA extraction method using phenol-chloroform (Peterson et al., 2009) requires careful liquid handling and a number of steps. Another method using silica columns, the RNeasy of Qiagen, provides easy and time-saving operation but is costly. In addition, the lysis solutions included in the kit do not contain phenol and, thus, are less effective in disrupting cells and tissues than phenol-containing lysis solutions. Recently, a new easy and time-saving method, Direct-zol (Sosanya et al., 2013), has been developed by combining these two methods. In the Direct-zol protocol, samples are lysed in phenol-containing solution and subsequently subjected to silica column-based RNA purification. However, the kit is costly (approximately 7 dollars per sample) for a large number of samples. From the viewpoint of high throughput, low cost, and robustness, the aforementioned methods are not feasible for large-scale sampling. Here, we introduce Direct-TRI, a high-throughput, cost-effective, and reliable RNA-extraction method using TRI Reagent-LS and 96-well silica column plate. This protocol consists of two steps: homogenization of samples in phenol-containing TRI Reagent-LS followed by the direct isolation of RNA from the phenol lysate with handmade washing solutions. Direct-TRI makes it possible to simultaneously process hundreds of RNA samples individually with a simple procedure and at an affordable cost. We show an example of a Direct-TRI application using whole zebrafish samples. Zebrafish is a model organism to study development, behavior, and disease in vertebrates (Goldsmith et al., 2012; Gore et al., 2018; Maeta et al., 2020). The large-scale gene expression analysis of zebrafish is a powerful tool for investigating the molecular basis underlying developmental events and diseases (Lee et al., 2020; Scholz, 2013). The combination of Direct-TRI and the cost-effective and high throughput RNA-Seq library preparation methods will make large-scale transcriptome analysis more useful not only for zebrafish samples but also for other organisms.