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RACE - Rapid Amplification of cDNA Ends02:35

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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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Updated: Apr 29, 2026

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Improved catalyzed reporter deposition, iCARD.

Jesper Lohse1, Kenneth Heesche Petersen, Nina Claire Woller

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Summary

Novel reporters enhance protein detection sensitivity and specificity. This new method allows for accurate in situ protein quantification in biological samples by counting dots, improving upon traditional assays.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunohistochemistry

Background:

  • Current protein detection methods can lack sensitivity or exhibit high background noise.
  • Accurate in situ protein quantification is crucial for understanding cellular processes and disease states.

Purpose of the Study:

  • To develop novel reporters for highly sensitive and specific in situ protein detection.
  • To establish a new method for protein quantification using image analysis of detected signals.

Main Methods:

  • Synthesis of novel reporters with extended hydrophilic linkers and polymerizing cross-linkers for efficient deposition.
  • Application of the reporters for staining HER2 proteins in cell lines and tissue samples.
  • Utilizing image analysis for counting detected protein signals (dots) for quantification.

Main Results:

  • The novel reporters demonstrated highly efficient deposition with virtually no background.
  • The method showed high specificity and sensitivity in detecting HER2 proteins in a cell line model.
  • Individual HER2 proteins were visualized as distinct dots in tissue, enabling quantification via image analysis.

Conclusions:

  • Developed novel reporters significantly improve the CARD (Catalyzed Reporter Deposition) method for sensitive in situ protein detection.
  • In situ protein quantification can be effectively achieved through object recognition and enumeration of dots, offering an alternative to intensity-based assays.