Yu-Chen Song, Mo-Xian Chen, Kai-Lu Zhang, Anireddy S. N. Reddy, Fu-Liang Cao, Fu-Yuan Zhu
{"title":"QuantAS: a comprehensive pipeline to study alternative splicing by absolute quantification of splice isoforms","authors":"Yu-Chen Song, Mo-Xian Chen, Kai-Lu Zhang, Anireddy S. N. Reddy, Fu-Liang Cao, Fu-Yuan Zhu","doi":"10.1111/nph.19193","DOIUrl":null,"url":null,"abstract":"<p>Alternative splicing (AS) is a mechanism by which cells generate abundant protein diversity from a limited number of genes (Baralle & Giudice, <span>2017</span>). AS plays a crucial role in regulating various life activities such as growth, development, and aging in plants (Zhu <i>et al</i>., <span>2017</span>; Godoy Herz & Kornblihtt, <span>2019</span>; Jabre <i>et al</i>., <span>2019</span>; Chen <i>et al</i>., <span>2020</span>; Reddy <i>et al</i>., <span>2020</span>; Zhang <i>et al</i>., <span>2020</span>), where it greatly influences plant growth, development, and response to biotic and abiotic stresses (Motion <i>et al</i>., <span>2015</span>; Laloum <i>et al</i>., <span>2018</span>; Chaudhary <i>et al</i>., <span>2019</span>; Chen <i>et al</i>., <span>2021</span>; Ganie & Reddy, <span>2021</span>; Saini <i>et al</i>., <span>2021</span>; Zhu <i>et al</i>., <span>2023</span>; Supporting Information Fig. S1). The traditional method for the identification of AS is semi-quantitative RT-PCR, which is easy to perform (Palusa <i>et al</i>., <span>2007</span>; Li <i>et al</i>., <span>2020</span>; Riegler <i>et al</i>., <span>2021</span>; Han <i>et al</i>., <span>2022</span>). Quantitative PCR (qPCR) is also widely used in AS research, as it enables real-time monitoring of fluorescence signals and accurate quantification of isoform copy numbers through the use of specific primers (Hefti <i>et al</i>., <span>2018</span>; Liu <i>et al</i>., <span>2018</span>; Huang <i>et al</i>., <span>2021</span>). With the emergence of digital PCR (dPCR), the identification methods of AS have become more diversified, which disperses each single target fragment into separate droplets as much as possible through the calculation of positive droplets (Fig. S2; Gao <i>et al</i>., <span>2021</span>).</p><p>Based on the urgent need for the accurate quantification of various isoforms, an AS detection method called QuantAS was established (Fig. 1), which allows us to accurately quantify all isoforms of genes based on absolute quantification technology and specific primer design. The method utilizes isoform-specific primers to overcome the isoform identification difficulty caused by different AS events and is designed by using the functional coding region as the isoform structure classification unit to ensure isoform independence (Fig. 2a). RT-qPCR enables real-time monitoring of changes in the fluorescence signal, quantification of differences between expression levels, and simultaneous detection of multiple in a single reaction. According to the copy number of different isoforms, isoform expression patterns can be identified by combining with absolute quantitative techniques. This method greatly increases the accuracy of identification and reduces the cost of repeated experiments. Furthermore, the absolute quantification of AS isoforms employing the combination of qPCR and dPCR could provide their respective advantages, thus rapidly obtaining all isoform information of the potential functional genes to be investigated.</p><p>QuantAS consists of three stages: (1) gene structure assembly and specific primer design, including AS event analysis; (2) accurate quantitative analysis of the isoforms in the treated samples using qPCR and dPCR to obtain the copy number of each isoform; and (3) absolute quantification, which involves data analysis to explore the existence and levels of isoforms (the outline of the protocol for QuantAS is shown in detail in Figs 1, S3).</p><p>In conclusion, QuantAS offers a universal method for the detection and quantification of isoforms in plants, allowing isoforms to be reclassified for different functional protein-coding regions and for precise AS identification to determine AS event types. QuantAS is also able to detect multiple isoforms simultaneously in a single reaction, thus decreasing the redundant identification and calculating the copy number of the complicated isoform according to MLE. Coupled with qPCR and dPCR techniques, it will allow rapid and precise screening of isoform changes that participate in physiological responses. The straightforward experimental design and procedures make QuantAS a valuable addition to the current tool chest for the study of AS, especially in the validation of splicing events identified in large-scale omics data.</p><p>None declared.</p><p>Y-CS wrote the manuscript and performed the experiments. M-XC and K-LZ performed the data analysis. ASNR and F-LC provided a critical review of the manuscript. F-YZ generally supervised the research group and designed the research.</p>","PeriodicalId":48887,"journal":{"name":"New Phytologist","volume":null,"pages":null},"PeriodicalIF":9.4000,"publicationDate":"2023-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/nph.19193","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"New Phytologist","FirstCategoryId":"99","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/nph.19193","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Agricultural and Biological Sciences","Score":null,"Total":0}
引用次数: 1
Abstract
Alternative splicing (AS) is a mechanism by which cells generate abundant protein diversity from a limited number of genes (Baralle & Giudice, 2017). AS plays a crucial role in regulating various life activities such as growth, development, and aging in plants (Zhu et al., 2017; Godoy Herz & Kornblihtt, 2019; Jabre et al., 2019; Chen et al., 2020; Reddy et al., 2020; Zhang et al., 2020), where it greatly influences plant growth, development, and response to biotic and abiotic stresses (Motion et al., 2015; Laloum et al., 2018; Chaudhary et al., 2019; Chen et al., 2021; Ganie & Reddy, 2021; Saini et al., 2021; Zhu et al., 2023; Supporting Information Fig. S1). The traditional method for the identification of AS is semi-quantitative RT-PCR, which is easy to perform (Palusa et al., 2007; Li et al., 2020; Riegler et al., 2021; Han et al., 2022). Quantitative PCR (qPCR) is also widely used in AS research, as it enables real-time monitoring of fluorescence signals and accurate quantification of isoform copy numbers through the use of specific primers (Hefti et al., 2018; Liu et al., 2018; Huang et al., 2021). With the emergence of digital PCR (dPCR), the identification methods of AS have become more diversified, which disperses each single target fragment into separate droplets as much as possible through the calculation of positive droplets (Fig. S2; Gao et al., 2021).
Based on the urgent need for the accurate quantification of various isoforms, an AS detection method called QuantAS was established (Fig. 1), which allows us to accurately quantify all isoforms of genes based on absolute quantification technology and specific primer design. The method utilizes isoform-specific primers to overcome the isoform identification difficulty caused by different AS events and is designed by using the functional coding region as the isoform structure classification unit to ensure isoform independence (Fig. 2a). RT-qPCR enables real-time monitoring of changes in the fluorescence signal, quantification of differences between expression levels, and simultaneous detection of multiple in a single reaction. According to the copy number of different isoforms, isoform expression patterns can be identified by combining with absolute quantitative techniques. This method greatly increases the accuracy of identification and reduces the cost of repeated experiments. Furthermore, the absolute quantification of AS isoforms employing the combination of qPCR and dPCR could provide their respective advantages, thus rapidly obtaining all isoform information of the potential functional genes to be investigated.
QuantAS consists of three stages: (1) gene structure assembly and specific primer design, including AS event analysis; (2) accurate quantitative analysis of the isoforms in the treated samples using qPCR and dPCR to obtain the copy number of each isoform; and (3) absolute quantification, which involves data analysis to explore the existence and levels of isoforms (the outline of the protocol for QuantAS is shown in detail in Figs 1, S3).
In conclusion, QuantAS offers a universal method for the detection and quantification of isoforms in plants, allowing isoforms to be reclassified for different functional protein-coding regions and for precise AS identification to determine AS event types. QuantAS is also able to detect multiple isoforms simultaneously in a single reaction, thus decreasing the redundant identification and calculating the copy number of the complicated isoform according to MLE. Coupled with qPCR and dPCR techniques, it will allow rapid and precise screening of isoform changes that participate in physiological responses. The straightforward experimental design and procedures make QuantAS a valuable addition to the current tool chest for the study of AS, especially in the validation of splicing events identified in large-scale omics data.
None declared.
Y-CS wrote the manuscript and performed the experiments. M-XC and K-LZ performed the data analysis. ASNR and F-LC provided a critical review of the manuscript. F-YZ generally supervised the research group and designed the research.
选择性剪接(AS)是细胞从有限数量的基因中产生丰富蛋白质多样性的一种机制(Baralle&;Giudice,2017)。AS在调节植物的生长、发育和衰老等各种生命活动中发挥着至关重要的作用(Zhu et al.,2017;Godoy-Herz和Kornblihtt,2019;Jabre et al.,2019;Chen et al.,2020;Reddy et al,以及对生物和非生物胁迫的反应(Motion等人,2015;Laloum等人,2018;Chaudhary等人,2019;Chen等人,2021;Ganie&;Reddy,2021;Saini等人,2021年;Zhu等人,2023;支持信息图S1)。鉴定AS的传统方法是半定量RT-PCR,其易于执行(Palusa等人,2007;李等人,2020;Riegler等人,2021;Han等人,2022)。定量PCR(qPCR)也广泛用于AS研究,因为它能够通过使用特异性引物实时监测荧光信号并准确定量同种型拷贝数(Hefti et al.,2018;刘等人,2018;Huang等人,2021)。随着数字PCR(dPCR)的出现,as的鉴定方法变得更加多样化,通过计算阳性液滴,将每个单个靶片段尽可能分散成单独的液滴(图S2;Gao等人,2021)。基于对各种异构体精确定量的迫切需要,建立了一种称为QuantAS的as检测方法(图1),这使我们能够基于绝对定量技术和特异性引物设计准确地量化所有基因的亚型。该方法利用异构体特异性引物来克服不同AS事件引起的异构体鉴定困难,并通过使用功能编码区作为异构体结构分类单元来设计,以确保异构体的独立性(图2a)。RT-qPCR能够实时监测荧光信号的变化,量化表达水平之间的差异,以及在单个反应中同时检测多个。根据不同亚型的拷贝数,结合绝对定量技术可以识别亚型的表达模式。这种方法大大提高了识别的准确性,降低了重复实验的成本。此外,使用qPCR和dPCR的组合对AS亚型进行绝对定量可以提供它们各自的优势,从而快速获得待研究的潜在功能基因的所有亚型信息。QuantAS包括三个阶段:(1)基因结构组装和特异性引物设计,包括AS事件分析;(2) 使用qPCR和dPCR对处理过的样品中的同种型进行精确定量分析,以获得每种同种型的拷贝数;和(3)绝对定量,包括数据分析,以探索异构体的存在和水平(QuantAS的方案大纲如图1、S3所示)。总之,QuantAS为检测和定量植物中的异构体提供了一种通用的方法,允许对不同功能蛋白编码区的同种型进行重新分类,并用于精确的AS鉴定以确定AS事件类型。QuantAS还能够在单个反应中同时检测多种异构体,从而减少冗余鉴定,并根据MLE计算复杂异构体的拷贝数。与qPCR和dPCR技术相结合,它将能够快速准确地筛选参与生理反应的同种型变化。简单的实验设计和程序使QuantAS成为当前AS研究工具箱的一个有价值的补充,特别是在验证大规模组学数据中确定的剪接事件方面。无声明。Y-CS编写了手稿并进行了实验。M-XC和K-LZ进行了数据分析。ASNR和F-LC对手稿进行了批评性评论。F-YZ对课题组进行了总体监督和设计。
期刊介绍:
New Phytologist is a leading publication that showcases exceptional and groundbreaking research in plant science and its practical applications. With a focus on five distinct sections - Physiology & Development, Environment, Interaction, Evolution, and Transformative Plant Biotechnology - the journal covers a wide array of topics ranging from cellular processes to the impact of global environmental changes. We encourage the use of interdisciplinary approaches, and our content is structured to reflect this. Our journal acknowledges the diverse techniques employed in plant science, including molecular and cell biology, functional genomics, modeling, and system-based approaches, across various subfields.