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Related Concept Videos

Experimental RNAi02:15

Experimental RNAi

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RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
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Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
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Searching Novel Clock Genes Using RNAi-Based Screening.

Bert Maier1, Stephan Lorenzen2,3, Anna-Marie Finger1

  • 1Laboratory of Chronobiology, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|December 7, 2020
PubMed
Summary
This summary is machine-generated.

This study introduces a method for RNA interference (RNAi) to find genes controlling circadian rhythms. The technique uses lentiviral delivery and live-cell recording, alongside new software for data analysis.

Keywords:
ChronoStarCircadian rhythmsLive-cell imagingLuciferase reporterRNA interferenceScreenTime-series analysisU-2 OS cells

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Area of Science:

  • Molecular Biology
  • Chronobiology
  • Genetics

Background:

  • RNA interference (RNAi) is a technique for gene silencing by targeting messenger RNA (mRNA).
  • High-throughput screening using RNAi is valuable for identifying genes involved in biological processes.
  • Understanding circadian rhythms is crucial for various physiological functions.

Purpose of the Study:

  • To develop a highly parallel RNA interference (RNAi) protocol for gene expression downregulation.
  • To identify novel components involved in the generation of circadian rhythms.
  • To present a new software tool for analyzing time-series data of circadian rhythms.

Main Methods:

  • Utilizing lentiviral gene transfer to deliver short hairpin RNA (shRNA) expressing plasmids for stable gene knockdown.
  • Employing live-cell bioluminescence recording to monitor circadian rhythms in synchronized reporter cells over multiple days.
  • Developing and applying the ChronoStar software for parallel time-series analysis of rhythm parameters.

Main Results:

  • Demonstrated efficient and stable gene knockdown in circadian reporter cells via lentiviral shRNA delivery.
  • Successfully monitored circadian rhythms using bioluminescence over extended periods.
  • Validated the ChronoStar software for accurate extraction of circadian rhythm parameters like period, phase, amplitude, and damping.

Conclusions:

  • The described RNAi protocol enables highly parallel gene downregulation for circadian rhythm research.
  • The combination of lentiviral shRNA, live-cell recording, and ChronoStar facilitates the discovery of circadian rhythm components.
  • This approach provides a robust platform for dissecting the genetic basis of biological clocks.