Experimental Design for Time-Series RNA-Seq Analysis of Gene Expression and Alternative Splicing

Nikoleta A. Tzioutziou, Allan B. James, Wenbin Guo, Cristiane P. G. Calixto, Runxuan Zhang, Hugh G. Nimmo, John W. S. Brown (Lead / Corresponding author)

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


RNA-sequencing (RNA-seq) is currently the method of choice for analysis of differential gene expression. To fully exploit the wealth of data generated from genome-wide transcriptomic approaches, the initial design of the experiment is of paramount importance. Biological rhythms in nature are pervasive and are driven by endogenous gene networks collectively known as circadian clocks. Measuring circadian gene expression requires time-course experiments which take into account time-of-day factors influencing variability in expression levels. We describe here an approach for characterizing diurnal changes in expression and alternative splicing for plants undergoing cooling. The method uses inexpensive everyday laboratory equipment and utilizes an RNA-seq application (3D RNA-seq) that can handle complex experimental designs and requires little or no prior bioinformatics expertise.

Original languageEnglish
Title of host publicationPlant Circadian Networks
Subtitle of host publicationMethods and Protocols
EditorsDorothee Staiger, Seth Davis, Amanda Melaragno Davis
Place of PublicationNew York
PublisherHumana Press
Number of pages16
ISBN (Electronic)9781071619124
ISBN (Print)9781071619117 , 9781071619148
Publication statusPublished - 2022

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Data validation
  • Experimental design
  • Primer design
  • RNA-seq
  • Time course

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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