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

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.