DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate
DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate
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DeLTa-Seq：从粗组织裂解液中直接裂解靶向 RNA-Seq
DOI：
10.1101/2020.09.15.299180
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Nagano,A.J.
中科院分区：
文献类型：
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作者：
Kashima;M.; Kamitani;M.;Nomura;Y.; Hirata;H.;Nagano,A.J.
基因表达的定量分析如RNA-Seq是研究各种生物现象的常用方法。尽管近十年来有了RNA- seq文库制备方法和测序平台的发展，但RNA提取仍然是各种生物组织样本RNA- seq中最费力和最昂贵的步骤。因此，在数千个样本中检测基因表达仍然很困难。结果我们开发的Direct-RT缓冲液可以在不提纯RNA的情况下进行组织样品的均质化和直接裂解反转录。在几种植物组织、酵母和斑马鱼幼虫的裂解物中，直接RT缓冲液中的DTT浓度可以阻止RNA降解，但不能阻止RT降解。这些裂解物在Direct- rt缓冲液中直接反转录产生的cDNA数量与纯化RNA合成的cDNA数量相当。为了最大限度地发挥Direct-RT缓冲液的优势，我们将Direct-RT和靶向RNA-Seq结合起来，开发了一种经济高效、高通量的定量方法，用于数百种基因的表达：DeLTa-Seq（直接裂解反转录和靶向RNA-Seq）。DeLTa-Seq方法可以大大提高基因表达分析的效率和准确性。对1056份拟南芥样品的δ - seq分析揭示了茉莉酸和水杨酸对拟南芥的温度依赖性。结论DeLTa-Seq方法可以实现对数千种动物、植物和微生物样品的大规模研究，如化学筛选、现场实验和关注个体差异的研究。此外，Direct-RT也有利于小组织的基因表达分析，因为小组织很难纯化足够的RNA进行实验。
BackgroundQuantification 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.ResultsHere, 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 inArabidopsis thaliana.ConclusionsThe 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.