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MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After...
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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
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Analysis of microRNA functions.

Carolyn Remsburg1, Kalin Konrad1, Nina Faye Sampilo1

  • 1Department of Biological Sciences, University of Delaware, Newark, DE, United States.

Methods in Cell Biology
|April 6, 2019
PubMed
Summary

Researchers can now validate microRNA (miRNA) targets and study their developmental roles in sea urchin embryos. This research provides new protocols for understanding gene regulation by miRNAs during development.

Keywords:
Dual luciferase assaysMicroRNAsMicroinjectionsPost-transcriptional regulationSea urchins

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Area of Science:

  • Developmental Biology
  • Molecular Biology
  • Genetics

Background:

  • MicroRNAs (miRNAs) are crucial regulators of gene expression, controlling mRNA translation and stability.
  • Post-transcriptional gene regulation by miRNAs is a conserved mechanism across diverse species.
  • Understanding miRNA function is key to deciphering complex biological pathways.

Purpose of the Study:

  • To present protocols for studying single miRNA function in sea urchin embryos.
  • To enable validation of direct miRNA targets and assessment of miRNA regulatory roles.
  • To investigate the impact of miRNA inhibition on developmental processes.

Main Methods:

  • Selection and design of effective miRNA inhibitors.
  • Experimental testing to identify direct miRNA targets.
  • Utilization of target protector morpholinos to assess miRNA inhibition effects.

Main Results:

  • Established methods for validating miRNA targets in sea urchin embryos.
  • Demonstrated techniques to evaluate the functional impact of miRNA inhibition.
  • Provided a framework for analyzing miRNA-mediated regulation in development.

Conclusions:

  • The described protocols empower researchers to dissect miRNA functions in sea urchin development.
  • These methods facilitate the understanding of how miRNAs regulate biological pathways.
  • This work contributes to the broader knowledge of miRNA roles in embryonic development.