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Related Concept Videos

Real Time RT-PCR02:57

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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...
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Related Experiment Video

Updated: Aug 30, 2025

Using High Content Imaging to Quantify Target Engagement in Adherent Cells
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Using High Content Imaging to Quantify Target Engagement in Adherent Cells

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Real-Time Cellular Thermal Shift Assay to Monitor Target Engagement.

Tino W Sanchez1, Michael H Ronzetti1, Ashley E Owens1

  • 1National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States.

ACS Chemical Biology
|September 1, 2022
PubMed
Summary
This summary is machine-generated.

We developed real-time cellular thermal shift assay (RT-CETSA) to improve drug discovery. This method efficiently detects ligand-target engagement in cells, advancing mechanism of action studies.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • Determining a molecule's mechanism of action is crucial for drug discovery.
  • Cellular thermal shift assay (CETSA) confirms target engagement but has throughput limitations.
  • Traditional CETSA requires individual samples per temperature, hindering optimization.

Purpose of the Study:

  • To develop a high-throughput method for assessing ligand-target engagement.
  • To introduce a real-time cellular thermal shift assay (RT-CETSA) platform.
  • To overcome the limitations of traditional CETSA for mechanism of action studies.

Main Methods:

  • Developed a real-time CETSA (RT-CETSA) platform by integrating a real-time PCR instrument with a CCD camera.
  • Engineered a thermally stable Nanoluciferase variant (ThermLuc) for monitoring target engagement.
  • Fused ThermLuc to diverse targets and tested with lactate dehydrogenase alpha inhibitors.

Main Results:

  • RT-CETSA captures a protein's full aggregation profile from a single sample.
  • Demonstrated significant correlation between RT-CETSA and enzymatic, biophysical, and cell-based assays.
  • Developed a data analysis pipeline to enhance RT-CETSA sensitivity for on-target binding detection.

Conclusions:

  • RT-CETSA technology significantly advances CETSA capabilities.
  • Facilitates efficient identification of ligand-target engagement in cells.
  • A critical step forward for assessing small molecule mechanism of action.