期刊
PLANT METHODS
卷 18, 期 1, 页码 -出版社
BMC
DOI: 10.1186/s13007-022-00930-x
关键词
Direct-lysate reverse transcription; Targeted RNA-Seq; Reducing reagent; Gene expression analysis
资金
- JST CREST [JPMJCR15O2]
- JST FOREST [JPMJFR210B]
- NEDO MOONSHOT Research & Development Program [JPNP18016]
A novel Direct-RT method was developed for homogenization and direct-lysate reverse transcription of tissue samples without the need for RNA purification. Integration of Direct-RT and targeted RNA-Seq led to the development of a cost-effective, high-throughput quantification method called DeLTa-Seq, which significantly improved the efficiency and accuracy of gene expression analysis. The DeLTa-Seq method demonstrated temperature-dependent effects in Arabidopsis thaliana.
Background Quantification of gene expression such as RNA-Seq is a popular approach to study various biological phenomena. Despite the development of RNA-Seq library preparation methods and sequencing platforms in the last decade, RNA extraction remains the most laborious and costly step in RNA-Seq of tissue samples of various organisms. Thus, it is still difficult to examine gene expression in thousands of samples. Results Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. The DTT concentration in Direct-RT buffer prevented RNA degradation but not RT in the lysates of several plant tissues, yeast, and zebrafish larvae. Direct reverse transcription on these lysates in Direct-RT buffer produced comparable amounts of cDNA to those synthesized from purified RNA. To maximize the advantage of the Direct-RT buffer, we integrated Direct-RT and targeted RNA-Seq to develop a cost-effective, high-throughput quantification method for the expressions of hundreds of genes: DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq). The DeLTa-Seq method could drastically improve the efficiency and accuracy of gene expression analysis. DeLTa-Seq analysis of 1056 samples revealed the temperature-dependent effects of jasmonic acid and salicylic acid in Arabidopsis thaliana. Conclusions The DeLTa-Seq method can realize large-scale studies using thousands of animal, plant, and microorganism samples, such as chemical screening, field experiments, and studies focusing on individual variability. In addition, Direct-RT is also beneficial for gene expression analysis in small tissues from which it is difficult to purify enough RNA for the experiments.
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