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Related Concept Videos

MicroRNAs01:22

MicroRNAs

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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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Optimized workflow to modify microRNA expression in primary human intravascular cells.

Safak Caglayan1, John-Bjarne Hansen2,3, Omri Snir2

  • 1Thrombosis Research Center (TREC), Institute of Clinical Medicine, UiT - The Arctic University of Norway, Tromsø, Norway. safak.caglayan@uit.no.

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|February 16, 2023
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Summary

Locked nucleic acid (LNA) miRNA inhibitors effectively reduce microRNA (miRNA) levels in primary cells, with LNA-PS inhibitors working without a lipid carrier. miRNA mimics require a lipid carrier for efficient cellular uptake and gene overexpression.

Keywords:
Endothelial cellGymnosisMonocyteTransfectionmiRNA inhibitormiRNA mimic

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Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Cellular Biology

Background:

  • MicroRNAs (miRNAs) are crucial for gene regulation and cell function.
  • Efficient manipulation of miRNA levels (knockdown or overexpression) is vital for research.
  • Commercial miRNA inhibitors and mimics vary in chemistry and require optimized transfection.

Purpose of the Study:

  • To investigate transfection conditions for miRNA inhibitors and mimics.
  • To assess the efficacy of two specific miRNAs (miR-15a-5p and miR-20b-5p) in human primary cells.
  • To compare different commercial miRNA products and delivery methods.

Main Methods:

  • Utilized mirVana and LNA miRNA inhibitors/mimics from two vendors.
  • Optimized transfection conditions in primary endothelial cells and monocytes.
  • Compared lipid-based (lipofectamine) delivery versus unassisted uptake.
  • Analyzed miRNA expression levels at 24 and 48 hours post-transfection.

Main Results:

  • LNA inhibitors (PE and PS) efficiently downregulated miR-15a-5p within 24 hours using a lipid carrier.
  • LNA-PS inhibitors achieved efficient miR-15a-5p knockdown without a lipid carrier.
  • MirVana inhibitors showed less efficiency, with no improvement after multiple transfections.
  • Both LNA and mirVana mimics demonstrated similar efficiency with a lipid carrier at 48 hours.
  • Neither mimic effectively induced overexpression without a lipid carrier.

Conclusions:

  • LNA miRNA inhibitors are highly effective for downregulating cellular miRNA expression.
  • LNA-PS miRNA inhibitors can be delivered effectively without lipid-based carriers.
  • MiRNA mimics necessitate lipid-based carriers for successful cellular uptake and overexpression.