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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
52.0K

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Real-time quantitative RT-PCR after laser-assisted cell picking

L Fink1, W Seeger, L Ermert

  • 1Department of Pathology, Justus-Liebig-Universität Giessen, Germany.

Nature Medicine
|November 11, 1998
PubMed
Summary

This study introduces a novel method combining laser-assisted cell picking and real-time PCR for precise mRNA quantitation in specific cells. The technique accurately measures TNF-alpha mRNA in rat lung macrophages, showing dose-dependent upregulation after endotoxin challenge.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Immunology

Background:

  • Quantifying gene expression in specific cell populations within intact tissues is challenging.
  • Traditional methods often require cell dissociation, potentially altering gene expression profiles.
  • Alveolar macrophages are key immune cells in the lung, and their transcriptional activity is crucial for understanding lung inflammation.

Purpose of the Study:

  • To develop and validate a technique for quantifying mRNA in a few cells isolated from intact organ structures.
  • To assess the expression of Tumor Necrosis Factor-alpha (TNF-alpha) mRNA in rat lung alveolar macrophages using the novel method.
  • To compare the results with a conventional cell isolation technique (bronchoalveolar lavage).

Main Methods:

  • Combining laser-assisted cell picking with real-time PCR for mRNA quantitation.
  • Microscopically controlled laser ablation of specific cells (alveolar macrophages) from stained lung tissue sections.
  • Utilizing Taq polymerase 5' nuclease activity and a dual-labeled fluorogenic probe for online fluorescence detection.
  • Employing a pseudogene-free sequence of porphobilinogen deaminase (PBGD) as an internal calibrator.
  • Stimulating rats with endotoxin and Interferon-gamma (IFN-gamma) to induce TNF-alpha expression.

Main Results:

  • Demonstrated a dose-dependent upregulation of TNF-alpha mRNA in alveolar macrophages from challenged rat lungs.
  • The ratio of TNF-alpha mRNA copies to PBGD mRNA copies increased significantly with endotoxin and IFN-gamma challenge.
  • Quantitative data from laser-picked cells closely matched data from parallel bronchoalveolar lavage samples.
  • The method successfully quantified mRNA in a small number of cells without RNA extraction, DNase digestion, or nested-PCR.

Conclusions:

  • The combined laser-assisted cell picking and real-time PCR technique is a powerful tool for quantifying mRNA in small, defined cell populations within intact organs.
  • This method allows for the assessment of transcriptional regulation in specific cell types, offering insights into cellular responses.
  • The technique minimizes sample loss and procedural steps, providing accurate and efficient gene expression analysis.