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Related Concept Videos

In-situ Hybridization02:31

In-situ Hybridization

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In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...
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Robust and sensitive in situ RNA detection using Yn-situ.

Yunming Wu1, Wenjing Xu1, Limei Ma1

  • 1Stowers Institute for Medical Research, Kansas City, MO 64110, USA.

Cell Reports Methods
|May 2, 2022
PubMed
Summary

We developed Yn-situ, a cost-effective method for highly sensitive RNA detection in tissues. This technique uses fewer probes for accurate quantification, improving upon existing hybridization chain reaction (HCR) methods.

Keywords:
Yn-situin situ hybridizationquantitative in situ hybridizationsingle-molecule RNA FISH

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • Accurate in situ detection of RNA in tissue sections is crucial for understanding gene expression.
  • Existing methods like hybridization chain reaction (HCR) and RNAscope can be probe-intensive and costly.

Purpose of the Study:

  • To develop a cost-effective, highly sensitive, and quantitative method for in situ RNA detection.
  • To improve upon existing HCR-based RNA detection techniques.

Main Methods:

  • Developed Yn-situ (Y-branched probe in situ hybridization), utilizing a single-strand DNA preamplifier and hybridization chain reaction (HCR).
  • Optimized an improved fixation step for enhanced RNA molecule detection.
  • Characterized method performance using fewer probes targeting short nucleotide sequences.

Main Results:

  • Yn-situ achieved high sensitivity, enabling RNA detection with fewer probes compared to traditional methods.
  • Demonstrated quantitative results with smaller puncta and a higher signal-to-noise ratio using only five probes.
  • Showcased the method's wide dynamic range for quantifying gene expression in olfactory sensory neurons.

Conclusions:

  • Yn-situ offers a sensitive, cost-effective, and quantitative approach for in situ RNA detection.
  • The method requires fewer probes and provides superior signal quality compared to existing HCR and RNAscope techniques.
  • Key steps are detailed to facilitate adoption in individual laboratories.