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Related Experiment Videos

Fluorescence in situ hybridization method for measuring transfection efficiency

L Cheng1, C D Bucana, Q Wei

  • 1University of Texas M.D. Anderson Cancer Center, Houston, USA.

Biotechniques
|September 1, 1996
PubMed
Summary

Fluorescence in situ hybridization (FISH) offers a sensitive method to measure transfection efficiency. This technique provides a direct assessment of transient expression vectors in cells, outperforming traditional cotransfection assays.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • Assessing transfection efficiency is crucial for gene expression studies.
  • Current methods like cotransfection with pCMV beta gal may not accurately reflect true transfection rates.
  • Transient expression vectors are widely used in research.

Purpose of the Study:

  • To evaluate fluorescence in situ hybridization (FISH) as a method for measuring transfection efficiency.
  • To compare FISH with cotransfection using pCMV beta gal for assessing transient expression vector pCMVcat in lymphoblasts and fibroblasts.

Main Methods:

  • Utilized fluorescence in situ hybridization (FISH) with a pCMVcat probe to visualize plasmid location post-transfection.
  • Employed the diethylaminoethyl-dextran method for transfecting pCMVcat into lymphoblasts and fibroblasts.

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  • Compared FISH results with those obtained from cotransfection using pCMV beta gal.

    • Main Results:

    • FISH demonstrated significantly higher transfection efficiencies compared to cotransfection.
    • Transfection efficiency was approximately 15 times greater in fibroblasts and 70 times greater in lymphoblasts when measured by FISH.
    • FISH allows direct visualization and quantification of plasmid localization.

    Conclusions:

    • FISH is a highly sensitive, specific, and direct method for measuring transfection efficiency of transient expression vectors.
    • The FISH method is valuable for evaluating laboratory assays, especially those focusing on quantitative transfection aspects and DNA damage effects.