High-Resolution RT-PCR Analysis of Alternative Barley Transcripts.

High-Resolution RT-PCR Analysis of Alternative Barley Transcripts. Methods Mol Biol. 2019;1900:269-281 Authors: Simpson CG, Fuller J, Rapazote-Flores P, Mayer CD, Calixto CPG, Milne L, Hedley PE, Booth C, Waugh R, Brown JWS 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. PMID: 30460571 [PubMed - in process]
Source: Mol Biol Cell - Category: Molecular Biology Authors: Tags: Methods Mol Biol Source Type: research