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In-situ Hybridization02:31

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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.
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Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
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Single-probe RNA FISH in Yeast.

Gable M Wadsworth1, Rasesh Y Parikh1, Harold D Kim1

  • 1School of Physics, Georgia Institute of Technology, Atlanta, GA, USA.

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|July 21, 2021
PubMed
Summary
This summary is machine-generated.

This study adapted RNA Fluorescence In Situ Hybridization (FISH) for single-probe quantification of mRNA in yeast. This method accurately measures short transcripts and reduces costs.

Keywords:
Fluorescence In Situ HybridizationBudding yeastRNA FISHSaccharomyces cerevisiaeSingle moleculeTranscription

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Quantitative profiling of mRNA expression is crucial for understanding cellular states.
  • Standard RNA Fluorescence In Situ Hybridization (FISH) uses multiple probes, limiting its ability to analyze certain RNA transcripts.
  • Challenges include short transcripts, splicing variations, and alternate initiation sites, which can make transcripts indistinguishable with multiple probes.

Purpose of the Study:

  • To adapt the standard FISH protocol for single-probe quantification of mRNA transcripts in budding yeast.
  • To enable accurate measurement of short transcripts or specific transcript features.
  • To reduce the cost associated with performing FISH experiments.

Main Methods:

  • Adaptation of the standard RNA Fluorescence In Situ Hybridization (FISH) protocol.
  • Utilized a single DNA oligonucleotide probe with a single fluorophore.
  • Applied the method to quantify transcripts within budding yeast cells.

Main Results:

  • Successfully adapted FISH for single-probe quantification of mRNA.
  • Enabled accurate quantification of short transcripts and specific transcript features.
  • Demonstrated a reduction in the cost of FISH analysis.

Conclusions:

  • The adapted single-probe FISH method provides a cost-effective and accurate approach for mRNA quantification in budding yeast.
  • This technique overcomes limitations of multi-probe FISH, particularly for analyzing short or complex transcripts.
  • Offers a valuable tool for molecular and cell biology research.