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Methodology for Subcellular Fractionation and MicroRNA Examination of Mitochondria, Mitochondria Associated ER Membrane (MAM), ER, and Cytosol from Human Brain.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2020; Vol. 2063, pp. 139-154. - Publication Year :
- 2020
-
Abstract
- Eukaryotic cell organelles exert unique functions individually but also interact with each other for essential cellular functions. This physical interface between the organelles serves as an important platform for biomolecule trafficking and signaling. Mitochondria are membrane-bound organelles and form a dynamic contact with other organelles. The interactions and communication between mitochondria and endoplasmic reticulum (ER) are facilitated by an ER specific domain, named mitochondria associated ER membrane (MAM). Due to its unique location, the MAM is a "hotspot" for important cell signaling and biochemical processes including calcium homeostasis, lipid synthesis/exchange, inflammasome and autophagosome formation, and mitochondria fission/fusion. Although techniques are available for isolation of organelle fractions including MAM, most utilize animal tissues and cell lines. Here we describe a protocol that is tailored to the isolation of highly purified MAM, mitochondria, ER, and cytosol from human brain. In addition, we include a protocol for the isolation of total RNA and subsequent analysis of microRNAs from these highly purified organelle fractions. Finally, we include a panel of protein markers that are useful for validating the enrichment and purity of each subcellular fraction.
- Subjects :
- Animals
Cytosol
Humans
MicroRNAs genetics
Mitochondria genetics
Neurodegenerative Diseases genetics
Neurodegenerative Diseases pathology
Polymerase Chain Reaction
Rats
Signal Transduction
Brain pathology
Endoplasmic Reticulum metabolism
MicroRNAs isolation & purification
Mitochondria metabolism
Mitochondrial Membranes metabolism
Subcellular Fractions metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 2063
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 31667768
- Full Text :
- https://doi.org/10.1007/978-1-0716-0138-9_11