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Related Concept Videos

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Riboswitches

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Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
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Nucleic acids are the most important macromolecules for the continuity of life. They carry the cell's genetic blueprint and carry instructions for its functioning.
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The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and a hydrogen on the deoxyribose's second carbon. The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms  a 5′ to 3′ phosphodiester linkage.
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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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Related Experiment Video

Updated: Oct 19, 2025

mRNA Interactome Capture from Plant Protoplasts
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Single Molecule RNA FISH in Arabidopsis Root Cells.

Susan Duncan1, Tjelvar S G Olsson2, Matthew Hartley2

  • 1Earlham Institute, Norwich Research Park, Norwich, United Kingdom.

Bio-Protocol
|September 20, 2021
PubMed
Summary

This study introduces a single molecule RNA fluorescence in situ hybridization (smFISH) protocol for precise mRNA detection and quantification in plant root cells. The method enables accurate localization of gene expression within fixed samples.

Keywords:
Fluorescent in situ hybridizationArabidopsisGene expressionSingle RNA moleculesTranscription

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Genetics

Background:

  • Gene expression regulation studies require advanced detection methods.
  • Accurate quantification and localization of mRNA are crucial for understanding gene function.
  • Existing in situ hybridization techniques have limitations in sensitivity and resolution.

Purpose of the Study:

  • To present a refined single molecule RNA fluorescence in situ hybridization (smFISH) protocol.
  • To enable the detection and quantification of individual mRNA molecules in plant root cells.
  • To facilitate accurate mRNA localization within fixed plant tissues.

Main Methods:

  • Adaptation of a single molecule RNA fluorescence in situ hybridization (smFISH) protocol.
  • Utilizing multiple (48) fluorescently labeled DNA oligonucleotides (20-mers) per transcript.
  • Employing a wide-field fluorescence microscope for signal detection.

Main Results:

  • The protocol allows for the detection of individual mRNA molecules.
  • Accurate quantification and spatial localization of mRNA are achievable in fixed plant root cells.
  • The method is adaptable to various genetic backgrounds.

Conclusions:

  • The presented smFISH protocol offers a robust and accessible method for studying gene expression.
  • This technique significantly enhances the ability to analyze mRNA dynamics in plant roots.
  • The simplicity and broad applicability make it valuable for plant research.