Most eukaryotic genes produce different transcripts of multiple isoforms by inclusion or exclusion of particular exons. The isoforms of a gene often play diverse functional roles, and thus it is necessary to accurately measure isoform expressions as well as gene expressions. While previous studies have demonstrated the strong agreement between mRNA sequencing (RNA-seq) and array-based gene and/or isoform quantification platforms (Microarray gene expression and Exon-array), the more recently developed NanoString platform has not been systematically evaluated and compared, especially in large-scale studies across different cancer domains. In this paper, we present a large-scale comparative study among RNA-seq, NanoString, array-based, and RT-qPCR platforms using 46 cancer cell lines across different cancer types. The goal is to understand and evaluate the calibers of the platforms for measuring gene and isoform expressions in cancer studies. We first performed NanoString experiments on 59 cancer cell lines with 404 custom-designed probes for measuring the expressions of 478 isoforms in 155 genes, and additional RT-qPCR experiments for a subset of the measured isoforms in 13 cell lines. We then combined the data with the matched RNA-seq, Exon-array, and Microarray data of 46 of the 59 cell lines for the comparative analysis. In the comparisons of the platforms for measuring the expressions at both isoform and gene levels, we found that (1) the agreement on isoform expressions is lower than the agreement on gene expressions across the four platforms; (2) NanoString and Exon-array are not consistent on isoform quantification even though both techniques are based on hybridization reactions; (3) RT-qPCR experiments are more consistent with RNA-seq and Exon-array than NanoString in isoform quantification; (4) different RNA-seq isoform quantification methods show varying estimation results, and among the methods, Net-RSTQ and eXpress are more consistent across the platforms; and (5) RNA-seq has the best overall consistency with the other platforms on gene expression quantification.

中文翻译：

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在四个平台上测量的同工型表达的大规模比较研究

大多数真核基因通过包含或排除特定外显子而产生多种同工型的不同转录本。基因的同工型经常发挥各种功能作用，因此必须准确测量同工型表达以及基因表达。尽管先前的研究表明mRNA测序（RNA-seq）与基于阵列的基因和/或同种型定量平台（微阵列基因表达和外显子阵列）之间有着很强的一致性，但尚未对较新开发的NanoString平台进行系统的评估和比较，尤其是在跨不同癌症领域的大规模研究中。在本文中，我们提出了使用46种不同癌细胞类型的癌细胞系进行RNA-seq，NanoString，基于阵列和RT-qPCR平台之间的大规模比较研究。目的是了解和评估癌症研究中用于测量基因和同工型表达的平台的能力。我们首先使用404个定制设计的探针在59个癌细胞系上进行了NanoString实验，以测量155个基因中478个同工型的表达，并针对13个细胞系中一部分测得的同工型进行了附加的RT-qPCR实验。然后，我们将数据与59个细胞系中46个细胞系的匹配的RNA-seq，外显子阵列和微阵列数据相结合，以进行比较分析。在用于测量同工型和基因水平的表达的平台的比较中，我们发现（1）在这四个平台上，同工型表达的一致性低于基因表达的一致性；（2）即使两种技术都基于杂交反应，NanoString和Exon阵列在同工型定量分析上也不一致。（3）RT-qPCR实验与RNA-seq和Exon-array相比在Nanoform异构体定量方面更一致；（4）不同的RNA-seq同工型定量方法显示出不同的估计结果，并且在这些方法中，Net-RSTQ和eXpress在平台之间更加一致；（5）RNA-seq在基因表达定量方面与其他平台具有最佳的整体一致性。