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Related Experiment Video

Updated: Feb 2, 2026

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

Published on: July 29, 2019

High-Resolution RT-PCR Analysis of Alternative Barley Transcripts.

Craig G Simpson¹, John Fuller², Paulo Rapazote-Flores³

¹Cell and Molecular Sciences, The James Hutton Institute, Dundee, Scotland, UK. Craig.Simpson@hutton.ac.uk.

Methods in Molecular Biology (Clifton, N.J.)

|November 22, 2018

Summary

A new high-resolution reverse transcription-PCR (HR RT-PCR) panel validates RNA-sequencing data for alternatively spliced transcripts. This method quantifies splice isoform proportions across various conditions and species.

Keywords:

Alternative splicing HR RT-PCR RNA-seq

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Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

The barley genome assembly and RNA-sequencing (RNA-seq) have revealed extensive alternative splicing.
Quantifying changes in splice isoform proportions is crucial for understanding gene expression regulation.

Purpose of the Study:

To introduce and detail a high-resolution reverse transcription-PCR (HR RT-PCR) based panel.
To validate alternatively spliced transcripts identified by RNA-seq.
To enable quantification of splice isoform proportion changes under diverse experimental conditions.

Main Methods:

Development of a high-resolution reverse transcription-PCR (HR RT-PCR) panel.
Validation of barley gene splice isoforms using HR RT-PCR.
Application of HR RT-PCR for quantifying splice isoform changes across conditions.

Main Results:

The HR RT-PCR panel accurately confirms alternatively spliced transcripts from RNA-seq data.
The method allows for precise quantification of splice isoform proportion shifts.
Validation of selected barley genes provides confidence in RNA-seq findings.

Conclusions:

The HR RT-PCR panel is a reliable tool for validating and quantifying alternative splicing events.
This assay can be broadly applied to any gene in any species for detailed transcript isoform analysis.
It enhances the confidence in genome-wide alternative splicing studies.