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Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy
Published on: June 19, 2017
Protocol for quantifying miRNA trafficking across the endosomal membrane
Syamantak Ghosh1, Kamalika Mukherjee2, Suvendra N Bhattacharyya3
1RNA Biology Research Laboratory, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
Abstract:
The import of miRNA across the endosomal membrane is an essential prerequisite for the loading of miRNAs into the endosomal lumen. These miRNAs are then incorporated into mature multivesicular bodies (MVBs), which then fuse with the plasma membrane, leading to the release of exosomes or extracellular vesicles carrying miRNAs into the intercellular space. Here, we present a reproducible in vitro assay for the quantification of miRNA import across the endosomal membrane. We detail the isolation of intact endosomes from mammalian cells, followed by the loading of synthetic, chemically modified microRNAs (miRNAs) into the vesicles in an RNase-resistant form. Integrating differential centrifugation and iodixanol density-gradient fractionation, we isolate endosomes free of endoplasmic reticulum contamination. Then, we use optimized in vitro cargo-loading techniques to facilitate subsequent assessment of miRNA uptake, stability, and delivery efficiency in the presence of altered external factors, such as ATP or substrate concentration. Ultimately, this protocol presents a standardized method for quantifying miRNA trafficking across the endosomal membrane.
Insights
This study introduces a novel assay to quantify microRNA (miRNA) import into endosomes. This method standardizes the measurement of miRNA trafficking essential for exosome formation and intercellular communication.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- MicroRNA (miRNA) import across the endosomal membrane is critical for their incorporation into extracellular vesicles.
- This process precedes the release of miRNAs into the intercellular space via exosomes.
Purpose of the Study:
- To develop and validate a reproducible in vitro assay for quantifying miRNA import across the endosomal membrane.
- To establish a standardized method for assessing miRNA trafficking and delivery efficiency.
Main Methods:
- Isolation of intact endosomes from mammalian cells.
- Loading of synthetic, chemically modified miRNAs into endosomes in an RNase-resistant form.
- Utilizing differential centrifugation and iodixanol density-gradient fractionation to purify endosomes, followed by optimized in vitro cargo-loading techniques.
Main Results:
- The protocol successfully isolates endosomes free from endoplasmic reticulum contamination.
- The assay allows for the assessment of miRNA uptake, stability, and delivery efficiency under varying external conditions (e.g., ATP concentration).
Conclusions:
- This standardized protocol provides a reliable method for quantifying miRNA transport across the endosomal membrane.
- The assay facilitates the study of factors influencing miRNA loading into extracellular vesicles.
