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Related Experiment Video

Updated: Jul 10, 2026

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition
11:48

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Published on: October 9, 2014

A sensitive procedure to detect alternatively spliced mRNA in pooled-tissue samples.

Germán Gastón Leparc1, Robi David Mitra

  • 1Department of Genetics and Center for Genome Sciences, Washington University in St Louis, 4444 Forest Park Parkway, Campus Box 8510, St Louis, MO 63108, USA.

Nucleic Acids Research
|November 15, 2007
PubMed
Summary

We developed a cost-effective method to validate alternative splicing predictions. This approach significantly increases sensitivity and specificity for detecting cassette exons, improving transcriptomic analysis.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Genomics aims to catalog all splice forms in the transcriptome.
  • Computational predictions of alternative splicing outpace experimental validation.
  • Efficient validation methods are crucial for accurate transcriptomic analysis.

Purpose of the Study:

  • To systematically evaluate methods for validating cassette exons.
  • To identify a cost-effective, sensitive, and specific validation protocol.
  • To improve the accuracy of alternative splicing prediction validation.

Main Methods:

  • Examined three reverse transcription (RT) priming methods: poly-dT, random, and pooled exon-specific.
  • Investigated two PCR amplification strategies: flanking PCR and semi-nested PCR.

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Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay
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Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

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A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA
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A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Published on: December 2, 2009

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Last Updated: Jul 10, 2026

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition
11:48

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Published on: October 9, 2014

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay
11:22

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Published on: August 26, 2018

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA
13:00

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Published on: December 2, 2009

  • Compared a novel method (pooled exon-specific RT + semi-nested PCR) against standard techniques.

    • Main Results:

    • The combined method of pooled exon-specific RT and semi-nested PCR achieved 97% detection sensitivity, compared to 14% for common methods.
    • The developed protocol demonstrated high specificity, with no false positives detected.
    • The method effectively validates alternative exons from whole-organism RNA in a single experiment.

    Conclusions:

    • A novel protocol using pooled exon-specific primers for RT and semi-nested PCR offers a highly sensitive and specific method for alternative exon validation.
    • This accurate and cost-effective approach addresses the critical need for validating computational predictions of alternative splicing.
    • The protocol enables comprehensive analysis of alternative splicing across multiple tissues efficiently.