Abstract
Assembly of the barley genome and extensive use of RNA-seq has resulted in an abundance of gene expression data and the recognition of wide-scale production of alternatively spliced transcripts. Here, we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) that confirms the accuracy of alternatively spliced transcripts from RNA-seq and allows quantification of changes in the proportion of splice isoforms between different experimental conditions, time points, tissues, genotypes, ecotypes, and treatments. By validating a selection of barley genes, use of the panel gives confidence or otherwise to the genome-wide global changes in alternatively spliced transcripts reported by RNA-seq. This simple assay can readily be applied to perform detailed transcript isoform analysis for any gene in any species.
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Acknowledgments
Work is supported by grants from the Biotechnology and Biological Sciences Research Council (BB/I00663X/1: to RW), and the Scottish Government Rural and Environment Science and Analytical Services division.
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Simpson, C.G. et al. (2019). High-Resolution RT-PCR Analysis of Alternative Barley Transcripts. In: Harwood, W. (eds) Barley. Methods in Molecular Biology, vol 1900. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8944-7_17
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DOI: https://doi.org/10.1007/978-1-4939-8944-7_17
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