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High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology.

Laetitia Chauve1, Jérémie Le Pen2, Francesca Hodge1

  • 1Babraham Institute.

Journal of Visualized Experiments : Jove
|June 16, 2020
PubMed
Summary

This study introduces a rapid, sensitive assay for measuring gene expression in Caenorhabditis elegans. The novel Worm-to-CT method enables high-throughput complementary DNA synthesis and quantitative PCR analysis from single or bulk samples.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Accurate gene expression analysis is crucial for understanding biological processes.
  • Existing methods for Caenorhabditis elegans gene expression analysis can be time-consuming and labor-intensive.
  • High-throughput methods are needed to accelerate research in model organisms.

Purpose of the Study:

  • To develop a fast, robust, and highly sensitive high-throughput assay for gene expression analysis in Caenorhabditis elegans.
  • To enable precise gene expression measurements from both single worms and bulk samples.
  • To integrate a novel complementary DNA (cDNA) preparation method with nanofluidic quantitative PCR (qPCR) technology.

Main Methods:

  • Development of the 'Worm-to-CT' protocol for direct cDNA synthesis from nematodes without mRNA isolation.
  • Utilizing nanofluidic quantitative PCR (qPCR) platforms for high-throughput reaction processing.
  • Evaluation of two nanofluidic chip formats: one supporting 9,216 reactions and another with 864 reactions.
  • Design of an extensive primer set for amplifying processed RNA from most C. elegans coding genes.

Main Results:

  • The Worm-to-CT method allows cDNA preparation from 96 worms in 3.5 hours, significantly increasing experimental throughput.
  • High-throughput RT-qPCR on nanofluidic chips enabled processing of thousands of reactions efficiently.
  • The assay successfully quantified mRNA levels of heat shock protein genes in single and bulk C. elegans samples.
  • Demonstrated the robustness and sensitivity of the developed assay for gene expression studies.

Conclusions:

  • The presented high-throughput RT-qPCR assay provides a rapid and sensitive tool for C. elegans gene expression studies.
  • The Worm-to-CT protocol streamlines cDNA synthesis, enhancing experimental efficiency.
  • This integrated approach facilitates precise gene expression measurements at single-worm and bulk levels, advancing C. elegans research.