High-Resolution RT-PCR Analysis of Alternative Barley Transcripts

Craig G. Simpson (Lead / Corresponding author), John Fuller, Paulo Rapazote-Flores, Claus Dieter Mayer, Cristiane P. G. Calixto, Linda Milne, Pete E. Hedley, Clare Booth, Robbie Waugh, John W. S. Brown

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

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.

Original languageEnglish
Title of host publicationBarley
Subtitle of host publicationMethods and Protocols
EditorsWendy A. Harwood
Place of PublicationNew York
PublisherHumana Press
Pages269-281
Number of pages13
Volume1900
ISBN (Electronic)9781493989447
ISBN (Print)9781493989423
DOIs
Publication statusPublished - 2019

Publication series

NameMethods in Molecular Biology
PublisherSpringer
ISSN (Print)1064-3745

Fingerprint

Hordeum
RNA
Polymerase Chain Reaction
Protein Isoforms
Genome
Ecotype
Genes
Reverse Transcription
Genotype
Gene Expression

Keywords

  • Alternative splicing
  • HR RT-PCR
  • RNA-seq

Cite this

Simpson, C. G., Fuller, J., Rapazote-Flores, P., Mayer, C. D., Calixto, C. P. G., Milne, L., ... Brown, J. W. S. (2019). High-Resolution RT-PCR Analysis of Alternative Barley Transcripts. In W. A. Harwood (Ed.), Barley: Methods and Protocols (Vol. 1900, pp. 269-281). (Methods in Molecular Biology). New York: Humana Press. https://doi.org/10.1007/978-1-4939-8944-7_17
Simpson, Craig G. ; Fuller, John ; Rapazote-Flores, Paulo ; Mayer, Claus Dieter ; Calixto, Cristiane P. G. ; Milne, Linda ; Hedley, Pete E. ; Booth, Clare ; Waugh, Robbie ; Brown, John W. S. / High-Resolution RT-PCR Analysis of Alternative Barley Transcripts. Barley: Methods and Protocols. editor / Wendy A. Harwood. Vol. 1900 New York : Humana Press, 2019. pp. 269-281 (Methods in Molecular Biology).
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Simpson, CG, Fuller, J, Rapazote-Flores, P, Mayer, CD, Calixto, CPG, Milne, L, Hedley, PE, Booth, C, Waugh, R & Brown, JWS 2019, High-Resolution RT-PCR Analysis of Alternative Barley Transcripts. in WA Harwood (ed.), Barley: Methods and Protocols. vol. 1900, Methods in Molecular Biology, Humana Press, New York, pp. 269-281. https://doi.org/10.1007/978-1-4939-8944-7_17

High-Resolution RT-PCR Analysis of Alternative Barley Transcripts. / Simpson, Craig G. (Lead / Corresponding author); Fuller, John; Rapazote-Flores, Paulo; Mayer, Claus Dieter; Calixto, Cristiane P. G.; Milne, Linda; Hedley, Pete E.; Booth, Clare; Waugh, Robbie; Brown, John W. S.

Barley: Methods and Protocols. ed. / Wendy A. Harwood. Vol. 1900 New York : Humana Press, 2019. p. 269-281 (Methods in Molecular Biology).

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

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AU - Waugh, Robbie

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N2 - 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.

AB - 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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Simpson CG, Fuller J, Rapazote-Flores P, Mayer CD, Calixto CPG, Milne L et al. High-Resolution RT-PCR Analysis of Alternative Barley Transcripts. In Harwood WA, editor, Barley: Methods and Protocols. Vol. 1900. New York: Humana Press. 2019. p. 269-281. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8944-7_17