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Related Experiment Video

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Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains
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Measuring mRNA Decay in Budding Yeast Using Single Molecule FISH.

Tatjana Trcek1, Samir Rahman2, Daniel Zenklusen2

  • 1Department of Cell Biology, Skirball Institute of Biomolecular Medicine, NYU School of Medicine, New York, NY, USA. tatjana.trcekpulisic@med.nyu.edu.

Methods in Molecular Biology (Clifton, N.J.)
|December 14, 2017
PubMed
Summary
This summary is machine-generated.

This study presents a new method using single-molecule fluorescent in situ hybridization (smFISH) to measure mRNA decay rates in individual yeast cells without perturbing cellular processes, enabling accurate mRNA turnover quantification.

Keywords:
Budding yeastFluorescent in situ hybridizationS. cerevisiaeSingle cellsSingle moleculeYeastmRNA decaymRNA decay ratemRNA half-lifemRNA turnoversmFISH

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Cellular messenger RNA (mRNA) levels are regulated by synthesis and decay rates.
  • Traditional mRNA decay measurements use chemical or genetic methods that can interfere with the process.
  • Single-molecule fluorescent in situ hybridization (smFISH) offers a way to measure decay in unperturbed single cells.

Purpose of the Study:

  • To provide a detailed protocol for quantifying mRNA decay in single Saccharomyces cerevisiae cells.
  • To enable accurate measurement of mRNA half-lives using smFISH in unperturbed cells.
  • To establish a method applicable to various cell types and tissues for mRNA turnover analysis.

Main Methods:

  • Utilizing single-molecule fluorescent in situ hybridization (smFISH) with DNA probes to label single cytoplasmic and nascent mRNAs.
  • Quantifying fluorescently labeled mRNAs in individual cells using automated spot detection algorithms.
  • Applying mathematical modeling to derive mRNA decay rates and half-lives from single-cell measurements.

Main Results:

  • A robust protocol for quantifying mRNA decay in single, unperturbed yeast cells was developed.
  • The method allows for the measurement of mRNA turnover for any specific mRNA.
  • Successful application of smFISH for precise mRNA decay rate determination in yeast.

Conclusions:

  • The developed smFISH protocol provides a valuable tool for studying mRNA decay dynamics in single cells.
  • This method overcomes limitations of population-based assays and chemical/genetic perturbations.
  • The approach is adaptable for studying mRNA turnover across diverse biological systems, including human tissues.