Advertisement

Barley pp 269-281 | Cite as

High-Resolution RT-PCR Analysis of Alternative Barley Transcripts

  • Craig G. SimpsonEmail 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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1900)

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.

Key words

HR RT-PCR Alternative splicing RNA-seq 

Notes

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.

References

  1. 1.
    Zhou HL, Luo G, Wise JA et al (2014) Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms. Nucleic Acids Res 42:701–713CrossRefGoogle Scholar
  2. 2.
    Reddy ASN, Marquez Y, Kalyna M et al (2013) Complexity of the alternative splicing landscape in plants. Plant Cell 25:3657–3683CrossRefGoogle Scholar
  3. 3.
    Naftelberg S, Schor IE, Ast G et al (2015) Regulation of alternative splicing through coupling with transcription and chromatin structure. Annu Rev Biochem 84:165–198CrossRefGoogle Scholar
  4. 4.
    Filichkin S, Priest HD, Megraw M et al (2015) Alternative splicing in plants: directing traffic at the crossroads of adaptation and environmental stress. Curr Opin Plant Biol 24:125–135CrossRefGoogle Scholar
  5. 5.
    Staiger D, Brown JWS (2013) Alternative splicing at the intersection of biological timing, development, and stress responses. Plant Cell 25:3640–3656CrossRefGoogle Scholar
  6. 6.
    Lee Y, Rio DC (2015) Mechanisms and regulation of alternative Pre-mRNA splicing. Annu Rev Biochem 84:291–323CrossRefGoogle Scholar
  7. 7.
    Mastrangelo AM, Marone D, Laidò G et al (2012) Alternative splicing: enhancing ability to cope with stress via transcriptome plasticity. Plant Sci 185–186:40–49CrossRefGoogle Scholar
  8. 8.
    Capovilla G, Pajoro A, Immink RG et al (2015) Role of alternative pre-mRNA splicing in temperature signaling. Curr Opin Plant Biol 27:97–103CrossRefGoogle Scholar
  9. 9.
    International Barley Sequencing Consortium (2012) A physical, genetic and functional sequence assembly of the barley genome. Nature 491:711–716CrossRefGoogle Scholar
  10. 10.
    Zhang Q, Zhang X, Pettolino F et al (2016) Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos. J Plant Physiol 191:127–139CrossRefGoogle Scholar
  11. 11.
    Zhang Q, Zhang X, Wang S et al (2016) Involvement of alternative splicing in barley seed germination. PLoS One 11:e0152824CrossRefGoogle Scholar
  12. 12.
    Simpson CG, Fuller J, Maronova M et al (2008) Monitoring changes in alternative precursor messenger RNA splicing in multiple gene transcripts. Plant J 53:1035–1048CrossRefGoogle Scholar
  13. 13.
    Marquez Y, Brown JWS, Simpson CG et al (2012) Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res 22:1184–1195CrossRefGoogle Scholar
  14. 14.
    James AB, Syed NH, Bordage S et al (2012) Alternative splicing mediates responses of the arabidopsis circadian clock to temperature changes. Plant Cell 24:961–981CrossRefGoogle Scholar
  15. 15.
    James A, Syed N, Brown J et al (2012) Thermoplasticity in the plant circadian clock: how plants tell the time-perature. Plant Signal Behav 7:1219–1223CrossRefGoogle Scholar
  16. 16.
    Raczynska KD, Simpson CG, Ciesiolka A et al (2010) Involvement of the nuclear cap-binding protein complex in alternative splicing in Arabidopsis thaliana. Nucleic Acids Res 38:265–278CrossRefGoogle Scholar
  17. 17.
    Streitner C, Köster T, Simpson CG et al (2012) An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with target transcripts in Arabidopsis thaliana. Nucleic Acids Res 40:11240–11255CrossRefGoogle Scholar
  18. 18.
    Simpson CG, Lewandowska D, Liney M et al (2014) Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially. New Phytol 203:424–436CrossRefGoogle Scholar
  19. 19.
    Calixto CP, Simpson CG, Waugh R et al (2016) Alternative splicing of barley clock genes in response to low temperature. PLoS One 11:e0168028CrossRefGoogle Scholar
  20. 20.
    Rundle SJ, Zielinski RE (1991) Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley. J Biol Chem 266:4677–4685PubMedGoogle Scholar
  21. 21.
    Milne I, Stephen G, Bayer M et al (2013) Using Tablet for visual exploration of second-generation sequencing data. Brief Bioinform 14:193–202CrossRefGoogle Scholar
  22. 22.
    Steijger T, Abril JF, Engström PG et al (2013) Assessment of transcript reconstruction methods for RNA-seq. Nat Methods 10:1177–1184CrossRefGoogle Scholar
  23. 23.
    Alamancos GP, Pagès A, Trincado JL et al (2015) Leveraging transcript quantification for fast computation of alternative splicing profiles. RNA 21:1521–1531CrossRefGoogle Scholar
  24. 24.
    Zhang R, Calixto CPG, Marquez Y et al (2016) AtRTD2: A Reference Transcript Dataset for accurate quantification of alternative splicing and expression changes in Arabidopsis thaliana RNA-seq data. bioRxiv.  https://doi.org/10.1101/051938
  25. 25.
    Brown JWS, Calixto CP, Zhang R (2017) High-quality reference transcript datasets hold the key to transcript-specific RNA-sequencing analysis in plants. New Phytol 213:525–530CrossRefGoogle Scholar
  26. 26.
    Kim SH, Koroleva OA, Lewandowska D et al (2009) Aberrant mRNA transcripts and the nonsense-mediated decay proteins UPF2 and UPF3 are enriched in the nucleolus. Plant Cell 21:2045–2057CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Craig G. Simpson
    • 1
    Email author
  • John Fuller
    • 1
  • Paulo Rapazote-Flores
    • 2
  • Claus-Dieter Mayer
    • 3
  • Cristiane P. G. Calixto
    • 4
  • Linda Milne
    • 2
  • Pete E. Hedley
    • 1
  • Clare Booth
    • 1
  • Robbie Waugh
    • 1
  • John W. S. Brown
    • 1
    • 4
  1. 1.Cell and Molecular SciencesThe James Hutton InstituteDundeeUK
  2. 2.Information and Computational SciencesThe James Hutton InstituteDundeeUK
  3. 3.Biomathematics and Statistics ScotlandDundeeUK
  4. 4.Division of Plant SciencesUniversity of Dundee at The James Hutton InstituteDundeeUK

Personalised recommendations