A large-scale comparative study of isoform expressions measured on four platforms

Wei Zhang,Rui Kuang,Jeongsik Yong,Raphael Petegrosso,Jiao Sun,Jeremy Chien,Jae-Woong Chang

doi:10.1186/s12864-020-6643-8

Abstract

BackgroundMost 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.ResultsIn 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.ConclusionIn 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.

Highlights

Most eukaryotic genes produce different transcripts of multiple isoforms by inclusion or exclusion of particular exons
We study the correlations of the expressions estimated by four different platforms including raw mRNA-Sequencing (RNA-seq), NanoString nCounter, Microarray/Exon-array and Quantitative reverse transcription polymerase chain reaction (RT-qPCR), at isoform and gene levels to better understand the characteristics of the estimations made by each platform
The raw mRNA-Sequencing (RNA-seq) data and Microarray expression data (HG-U133 Plus 2.0 Array) of the 46 cancer cell lines were downloaded from Cell Line Encyclopedia (CCLE) and processed for comparison with the NanoString nCounter data

Summary

Introduction

Most eukaryotic genes produce different transcripts of multiple isoforms by inclusion or exclusion of particular exons. 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. Recent studies have estimated that alternative splicing events exist in more than 95% of multi-exon genes in human and mouse [1, 2], and the mechanism provides the opportunity to create protein isoforms of differing functions from a single gene in a cellular system. Several high-throughput platforms have been developed during the last decades for transcriptome studies, including mRNA sequencing (RNA-seq), array-based technologies (Microarray and Exon-arrays), quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the more recently developed NanoString’s nCounter technology (Fig. 1). Different platforms can report inconsistent isoform expressions measured on the RNAs extracted from the same cell sample

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Conclusion