Elsevier

Methods

Volume 37, Issue 4, December 2005, Pages 345-359
Methods

Detection and measurement of alternative splicing using splicing-sensitive microarrays

https://doi.org/10.1016/j.ymeth.2005.09.007Get rights and content

Abstract

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.

Introduction

Splicing is required for proper expression of the vast majority of eukaryotic genes. Without information about splicing, prediction of protein coding potential of a eukaryotic genome is difficult if not impossible [1]. Compounding the problem, different mRNAs from the same gene (mRNA isoforms or splice variants) can be produced, encoding related proteins with distinct functions [2], [3]. Alternative splicing is a major source of protein diversity in higher eukaryotes, expanding the coding potential of the genome [2], [4], [5], [6]. There are few methods available to most researchers that allow large-scale changes in splicing of many genes to be detected or measured in a single experiment.

The use of microarrays has transformed the way gene expression is studied in a wide variety of eukaryotic systems. Microarrays can screen for the presence of mRNA from many genes in many defined samples, allowing the experimentalist to match changes in the landscape of gene activity to other biological events [7], [8], [9]. Unfortunately, standard microarray designs that strive to measure only the overall level of mRNA from each gene provide little or no information about splicing. Standard methods of splicing analysis such as reverse transcriptase primer extension, nuclease protection mapping, and cDNA cloning are too laborious to scale up for analysis of large numbers of genes and samples. To more generally determine the contribution of alternative splicing to programs of gene expression, parallel methods for measuring splicing en masse have been developed.

Several groups have contributed to the development of parallel methods for analyzing splicing. One method is to use arrays of spotted oligonucleotides designed to discriminate different RNAs from the same gene by targeting the distinct sequence features that characterize their differential splicing. Oligonucleotide probes that span splice junctions sample the presence and amount of RNA joined by particular splicing events. Other oligonucleotide probes are placed within exons in order to determine individual exon levels as well as overall mRNA level [10], [11]. Recent efforts using both the Affymetrix platform [12], [13] and the Rosetta platform [14], [15], [16], [17], [18] have shown promise in the application of this approach to alternative splicing. A second method for detecting the joining of specific exons provides for parallel measurement of many splicing events in the same experiment by using the spliced RNA as a template for the ligation of oligonucleotides. Ligation of specific pairs of oligonucleotides creates a PCR replicon that is detected after amplification, and reveals which exons are joined with which in the sample [19].

Here we describe the design, application, and analysis of simple printed oligonucleotide microarrays to the parallel detection and measurement of alternative splicing. The first section of this article discusses theoretical aspects of the problem. In the second section, we present two simple contexts in which to test and illuminate the principles and issues. First, we use a modified two-intron yeast gene altered in the branch point of the first intron that gives rise to different amounts of alternatively spliced RNAs to show that specificity of the array elements for a given isoform depends as expected on oligonucleotide probe length and hybridization temperature. Second, we identify and validate differential alternative splicing in human cancer cell lines using a small array carrying probes for 64 genes. Well-designed splicing-sensitive microarrays can be broadly applied in small scale or on genomic scale for parallel detection of alternative splicing in a flexible, cost-effective fashion.

Section snippets

Alternative splicing and gene models

To detect and measure alternatively processed RNAs requires a hypothesis or model describing the structures of the RNAs that need to be distinguished. Alignment of the sequences of mature RNAs to the gene sequence allows identification of exon boundaries and splice junctions. A common result is called exon skipping, wherein two alternative mRNA forms are produced that differ by the presence of a single exon (Fig. 1A). Since alternative mRNAs are formed, alternative exons and junctions (or their

A modified DYN2 test gene

The DYN2 gene is one of several known multi-intron genes in the genome of Saccharomyces cerevisiae. Normally, the second exon of DYN2 is included, and only a very small amount of mRNA that skips this exon can be detected [40]. Mutations in or near the first intron branchpoint stimulate skipping of the second exon and to a lesser extent allow retention of the first intron (Fig. 4A [40]). Severe branchpoint mutations prevent formation of the exon 2 included form, converting the mRNA population

Conclusions

The application of microarrays to splicing is still in a stage of development. The early experiments are promising, and it is very clear that for a large majority of genes and splicing events, microarrays will operate well to detect splicing changes. The numbers of genes and splicing events that can be accessed in a single experiment approaches a hundred to a thousand fold more than can reasonably be accessed using RT-PCR, primer extension, or nuclease protection experiments. This huge increase

Acknowledgments

Funding for the development of splicing-sensitive microarrays in our laboratory has been generously provided by the University of California Cancer Research Coordinating Committee, the Packard Foundation, the W.M. Keck Foundation (through its support of the RNA Center at UCSC), NIH Grant R01 GM 040478 to M. Ares, and R24 GM 070857 to D. Black, M. Ares, and X.-D. Fu. Support for the UCSC Microarray Facility also comes from NHGRI P41 HG02371 to David Haussler. Funding for students in the Hughes

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