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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Nonsense-mediated mRNA Decay02:27

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The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
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Related Experiment Video

Updated: Oct 6, 2025

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains
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Measuring mRNA Decay with Roadblock-qPCR.

Maegan J Watson1, Carson C Thoreen1

  • 1Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut.

Current Protocols
|January 18, 2022
PubMed
Summary
This summary is machine-generated.

Roadblock-qPCR is a new method to measure mRNA decay kinetics in living cells. This technique avoids transcription shutoff, offering a more accurate way to study gene regulation and function.

Keywords:
4-thiouridinemRNA stabilityqPCR

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Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Post-transcriptional Modification

Background:

  • mRNA stability is crucial for gene regulation, but measuring mRNA half-lives is challenging.
  • Existing methods like transcription shutoff perturb cell function and gene expression.
  • Understanding mRNA decay provides key insights into gene function.

Purpose of the Study:

  • To introduce Roadblock-qPCR, a simple, economical, and quick method for measuring mRNA decay kinetics in living cells.
  • To provide an alternative to transcription shutoff strategies, avoiding indirect perturbation of gene expression.
  • To enable accurate measurement of endogenous mRNA half-lives.

Main Methods:

  • Cells are incubated with 4-thiouridine (4sU), incorporated into nascent mRNA.
  • RNA is extracted and treated with N-ethylmaleimide (NEM) to modify 4sU.
  • NEM-modified 4sU acts as a roadblock, depleting nascent transcripts from cDNA pool for qPCR analysis of pre-existing mRNA decay.

Main Results:

  • Roadblock-qPCR allows for the monitoring of decay rates of non-4sU-labeled pre-existing mRNAs.
  • The method accurately measures half-lives of endogenous mRNAs with varying stabilities.
  • Spike-in standards enhance the efficiency and accuracy of the measurements.

Conclusions:

  • Roadblock-qPCR is an effective method for measuring mRNA decay kinetics without affecting cell viability.
  • This technique offers a valuable tool for studying gene regulation and function.
  • The method avoids artifacts associated with transcription shutoff strategies, providing more reliable data.