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Related Experiment Videos

High-throughput RT-PCR analysis of multiple transcripts using a microplate RNA isolation procedure

S Su1, R G Vivier, M C Dickson

  • 1Glaxo Wellcome Research and Development, Stevenage, Herts, UK.

Biotechniques
|June 1, 1997
PubMed
Summary

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A novel high-throughput reverse transcription PCR (RTPCR) assay enables sensitive RNA transcript analysis from small cell samples. This method is compatible with drug screening solvents and identifies gene expression changes and inhibitors.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Pharmacology

Background:

  • Analyzing low-copy cellular RNA transcripts requires sensitive and high-throughput methods.
  • Existing assays may be incompatible with solvents used in drug screening.
  • Investigating differential gene expression in immune cells is crucial for understanding disease and drug responses.

Purpose of the Study:

  • To develop a high-throughput, multiplex reverse transcription PCR (RTPCR) assay for analyzing low-copy cellular RNA transcripts.
  • To ensure the assay's compatibility with solvents commonly used in high-throughput drug screening.
  • To apply the assay for studying differential mRNA expression and identifying transcriptional inhibitors.

Main Methods:

  • Developed a microplate-based RNA extraction and RTPCR protocol for high throughput.

Related Experiment Videos

  • Utilized multiwelled agarose gel electrophoresis with SYBR Green I dye for PCR product visualization.
  • Validated the assay's performance with solvents like dimethyl sulfoxide and methanol.

    • Main Results:

    • The assay successfully analyzed medium-to low-copy cellular RNA transcripts from small cell numbers (10^4).
    • Transcriptional analysis remained unaffected by drug screening solvents at required concentrations.
    • Demonstrated successful application in analyzing differential mRNA expression of tumor necrosis factor alpha (TNF-alpha) and Interleukin-1 beta (IL-1 beta) in lipopolysaccharide (LPS)-stimulated THP-1 cells.
    • Identified specific inhibitors of IL-1 beta transcription.

    Conclusions:

    • The developed high-throughput multiplex RTPCR assay is a robust tool for sensitive RNA transcript analysis.
    • The assay is suitable for applications in drug discovery and studying gene expression in limited cell samples.
    • This method facilitates the investigation of inflammatory responses and the identification of therapeutic targets.