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

Abstract

Abstract Background 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. Results 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. Conclusion 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.

Keywords

• Isoform-level expression
• Cross-platform comparison
• NanoString
• RNA-seq
• Exon-array
• Microarray gene expression