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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.
|Title of host publication||Barley|
|Subtitle of host publication||Methods and Protocols|
|Editors||Wendy A. Harwood|
|Place of Publication||New York|
|Number of pages||13|
|Publication status||Published - 2019|
|Name||Methods in Molecular Biology|
- Alternative splicing
- HR RT-PCR
ASJC Scopus subject areas
- Molecular Biology
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