High-Resolution RT-PCR Analysis of Alternative Barley Transcripts

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 RNAseq 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.

Alternative Splicing of Circadian Clock Genes Correlates With Temperature in Field-Grown Sugarcane

“…High-Resolution RT-PCR (HR RT-PCR) reactions were performed based on Simpson et al (2007) and Simpson et al (2019). For all reactions, the forward primer was labeled with 6carboxyfluorescein (FAM).…”

“…We used HR RT-PCR (Simpson et al, 2019) to examine the daily dynamics of the expressed isoforms of the sugarcane circadian clock genes in two different seasons, winter and summer, using field-grown plants that were 4 and 9 months old, respectively. Briefly, the HR RT-PCR system uses fluorescently labelled primers to amplify across an AS event, followed by fragment analysis in an automatic DNA sequencer that quantifies the relative levels of RT-PCR products and thereby splicing ratios that reflect different splice site choices.…”