Methodology for Subcellular Fractionation and MicroRNA Examination of Mitochondria, Mitochondria Associated ER Membrane (MAM), ER, and Cytosol from Human Brain

Paresh Prajapati, Wang Xia Wang, Peter T. Nelson, Joe E. Springer

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

5 Scopus citations

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Pages139-154
Number of pages16
DOIs
StatePublished - 2020

Publication series

NameMethods in Molecular Biology
Volume2063
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Publisher Copyright:
© Springer Science+Business Media, LLC, part of Springer Nature 2020.

Keywords

  • Human brain
  • MicroRNA
  • Mitochondria associated ER membrane (MAM)
  • Neurodegenerative diseases
  • RT-qPCR
  • Subcellular fractionation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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