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MISSION esiRNA for RNAi Screening in Mammalian Cells
Published on: May 12, 2010
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Optimizing miRNA transfection for screening in precision cut lung slices.
Joanna Nowakowska1,2, Nika Gvazava3,4,5,6, Wojciech Langwiński1
1Molecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical Sciences, Poznan, Poland.
American Journal of Physiology. Lung Cellular and Molecular Physiology
|September 10, 2024
Summary
We developed a lipid nanoparticle (LNP) protocol for microRNA (miRNA) transfection in precision cut lung slices (PCLS). This method enables efficient miRNA delivery and visualization in 3-D lung models for drug screening.
Area of Science:
- * Lung tissue engineering and 3D modeling
- * RNA-based therapeutics and drug delivery
- * High-content imaging and screening
Background:
- * Precision cut lung slices (PCLS) are advanced 3D lung models preserving native microenvironments.
- * Studying microRNA (miRNA) effects in PCLS is crucial for disease research and therapeutic development.
- * Current miRNA transfection methods for PCLS require custom functionalization, limiting high-throughput screening.
Purpose of the Study:
- * To develop and optimize a lipid nanoparticle (LNP)-based protocol for miRNA transfection in PCLS.
- * To enable higher-throughput screening of miRNA sequences and their effects.
- * To establish a reproducible method for miRNA delivery in a complex 3D lung model.
Main Methods:
- * Generation of 4 mm PCLS from rodent lungs using a vibratome.
- * Evaluation of six commercially available LNPs for miRNA transfection efficacy using fluorescently labeled miRNA (TYE665).
- * Assessment of transfection efficiency via live and confocal fluorescence microscopy, and cytotoxicity via WST-1 and LDH assays.
Main Results:
- * All tested LNPs facilitated miRNA transfection in PCLS without significant cytotoxicity or metabolic impairment.
- * RNAiMAX and INTERFERin LNPs showed enhanced transfection efficacy, with detection at concentrations as low as 25 nM.
- * Cellular membrane permeability of PCLS was confirmed to be highest within the initial 24 hours post-slicing.
Conclusions:
- * LNP-based miRNA transfection is feasible and effective in PCLS.
- * The developed protocol allows visualization of miRNA delivery in live or fixed PCLS.
- * This method supports future high-throughput screening of diverse miRNA sequences in a relevant 3D lung model.
