High-Resolution RT-PCR Analysis of Alternative Barley Transcripts

Craig G. Simpson; 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

doi:10.1007/978-1-4939-8944-7_17

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

Plant Sciences

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

2 Citations (Scopus)

278 Downloads (Pure)

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 language	English
Title of host publication	Barley
Subtitle of host publication	Methods and Protocols
Editors	Wendy A. Harwood
Place of Publication	New York
Publisher	Humana Press
Pages	269-281
Number of pages	13
Volume	1900
ISBN (Electronic)	9781493989447
ISBN (Print)	9781493989423
DOIs	https://doi.org/10.1007/978-1-4939-8944-7_17
Publication status	Published - 2019

Publication series

Name	Methods in Molecular Biology
Publisher	Springer
ISSN (Print)	1064-3745

Keywords

Alternative splicing
HR RT-PCR
RNA-seq

ASJC Scopus subject areas

Molecular Biology
Genetics

Access to Document

10.1007/978-1-4939-8944-7_17

Author Accepted ManuscriptAccepted author manuscript, 448 KB

Cite this

Simpson, C. G., Fuller, J., Rapazote-Flores, P., Mayer, C. D., Calixto, C. P. G., Milne, L., Hedley, P. E., Booth, C., Waugh, R., & 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). Humana Press. https://doi.org/10.1007/978-1-4939-8944-7_17

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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.",

keywords = "Alternative splicing, HR RT-PCR, RNA-seq",

author = "Simpson, {Craig G.} and John Fuller and Paulo Rapazote-Flores and Mayer, {Claus Dieter} and Calixto, {Cristiane P. G.} and Linda Milne and Hedley, {Pete E.} and Clare Booth and Robbie Waugh and Brown, {John W. S.}",

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year = "2019",

doi = "10.1007/978-1-4939-8944-7_17",

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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 et al.
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 proceeding › Chapter (peer-reviewed) › peer-review

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AU - Rapazote-Flores, Paulo

AU - Mayer, Claus Dieter

AU - Calixto, Cristiane P. G.

AU - Milne, Linda

AU - Hedley, Pete E.

AU - Booth, Clare

AU - Waugh, Robbie

AU - Brown, John W. S.

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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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High-Resolution RT-PCR Analysis of Alternative Barley Transcripts

Abstract

Publication series

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

A Reference Transcript Database for Improved Analysis of RNA-seq Data from Barley

16 ERA-CAPS Barley Yield Associated Networks (Joint with IPK Gatersleben as lead and University of Minnesota)

Cite this

High-Resolution RT-PCR Analysis of Alternative Barley Transcripts

Abstract

Publication series

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Projects

A Reference Transcript Database for Improved Analysis of RNA-seq Data from Barley

16 ERA-CAPS Barley Yield Associated Networks (Joint with IPK Gatersleben as lead and University of Minnesota)

Cite this