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Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.

Harry M T Choi1, Maayan Schwarzkopf1, Mark E Fornace2

  • 1Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

Development (Cambridge, England)
|June 28, 2018
PubMed
Summary
This summary is machine-generated.

Third-generation in situ hybridization chain reaction (HCR) imaging offers enhanced mRNA expression analysis. This advanced method provides automatic background suppression for improved accuracy and versatility across diverse organisms and applications.

Keywords:
Automatic background suppressionIn situ HCR v3.0Multiplexed quantitative in situ hybridizationdHCR imagingqHCR flow cytometryqHCR imaging

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • Traditional in situ hybridization methods faced challenges in imaging mRNA expression, limiting multiplexing, quantitation, sensitivity, and resolution.
  • Hybridization chain reaction (HCR) based in situ hybridization has previously addressed some of these limitations.
  • Further improvements were needed for enhanced robustness and ease of use across various biological systems.

Purpose of the Study:

  • To introduce third-generation in situ HCR (HCR v3.0) with automatic background suppression.
  • To augment the capabilities of HCR for mRNA expression analysis, enhancing signal-to-background ratio and simplifying probe set optimization.
  • To enable advanced quantitative analysis modes for diverse biological samples.

Main Methods:

  • Development of third-generation HCR probes and amplifiers with integrated automatic background suppression.
  • Implementation of three distinct quantitative analysis modes: qHCR imaging, qHCR flow cytometry, and dHCR imaging.
  • Application of HCR v3.0 in whole-mount vertebrate embryos, mammalian cells, bacterial cells, and thick autofluorescent samples.

Main Results:

  • HCR v3.0 demonstrates automatic background suppression, preventing reagent-generated amplified background.
  • Achieved a higher signal-to-background ratio, enhancing performance and robustness.
  • Enabled three multiplexed quantitative analysis modes: analog relative quantitation (qHCR imaging and flow cytometry) and digital absolute quantitation (dHCR imaging).

Conclusions:

  • HCR v3.0 significantly advances in situ mRNA expression analysis by providing automatic background suppression.
  • The enhanced method offers improved accuracy, robustness, and versatility for imaging mRNA in diverse organisms and sample types.
  • HCR v3.0 facilitates high-throughput expression profiling and precise molecular quantitation, expanding applications in biological research.