Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2024 Nov 15

PMID 39546267

Authors

Julian Geiger

Louise T Dalgaard

Affiliations

Soon will be listed here.

Abstract

The presence of non-coding RNAs, such as microRNAs (miRNAs), in mitochondria has been reported by several studies. The biological roles and functions of these mitochondrial miRNAs ("mitomiRs") have not been sufficiently characterized, but the mitochondrial localization of miRNAs has recently gained significance due to modified mitomiR-populations in certain states of diseases. Here, we describe the isolation and analysis of mitochondrial RNAs from rat liver tissue and HepG2 cells. The principle of the analysis is to prepare mitochondria by differential centrifugation. Cytosolic RNA contamination is eliminated by RNase A treatment followed by percoll gradient-purification and RNA-extraction. Small RNA content is verified by capillary electrophoresis. Mitochondrial miRNAs are detected by Q-PCR following synthesis of cDNA. After Q-PCR based mitomiR-profiling, the Normfinder algorithm is applied to identify suitable reference miRNAs to use as normalizers for mitochondrial input and data analysis. The described procedure depicts a simple way of isolating and quantifying mitomiRs in tissue and cell culture samples.

References

Ro S, Ma H, Park C, Ortogero N, Song R, Hennig G . The mitochondrial genome encodes abundant small noncoding RNAs. Cell Res. 2013; 23(6):759-74. PMC: 3674384. DOI: 10.1038/cr.2013.37. View

Bandiera S, Ruberg S, Girard M, Cagnard N, Hanein S, Chretien D . Nuclear outsourcing of RNA interference components to human mitochondria. PLoS One. 2011; 6(6):e20746. PMC: 3113838. DOI: 10.1371/journal.pone.0020746. View

Geiger J, Dalgaard L . Interplay of mitochondrial metabolism and microRNAs. Cell Mol Life Sci. 2016; 74(4):631-646. PMC: 11107739. DOI: 10.1007/s00018-016-2342-7. View

Kren B, Wong P, Sarver A, Zhang X, Zeng Y, Steer C . MicroRNAs identified in highly purified liver-derived mitochondria may play a role in apoptosis. RNA Biol. 2008; 6(1):65-72. PMC: 3972804. DOI: 10.4161/rna.6.1.7534. View

Rackham O, Shearwood A, Mercer T, Davies S, Mattick J, Filipovska A . Long noncoding RNAs are generated from the mitochondrial genome and regulated by nuclear-encoded proteins. RNA. 2011; 17(12):2085-93. PMC: 3222122. DOI: 10.1261/rna.029405.111. View

Bandiera S, Mategot R, Girard M, Demongeot J, Henrion-Caude A . MitomiRs delineating the intracellular localization of microRNAs at mitochondria. Free Radic Biol Med. 2013; 64:12-9. DOI: 10.1016/j.freeradbiomed.2013.06.013. View

Tarassov I, Kamenski P, Kolesnikova O, Karicheva O, Martin R, Krasheninnikov I . Import of nuclear DNA-encoded RNAs into mitochondria and mitochondrial translation. Cell Cycle. 2007; 6(20):2473-7. DOI: 10.4161/cc.6.20.4783. View

Wang W, Visavadiya N, Pandya J, Nelson P, Sullivan P, Springer J . Mitochondria-associated microRNAs in rat hippocampus following traumatic brain injury. Exp Neurol. 2015; 265:84-93. PMC: 4346439. DOI: 10.1016/j.expneurol.2014.12.018. View

Jagannathan R, Thapa D, Nichols C, Shepherd D, Stricker J, Croston T . Translational Regulation of the Mitochondrial Genome Following Redistribution of Mitochondrial MicroRNA in the Diabetic Heart. Circ Cardiovasc Genet. 2015; 8(6):785-802. PMC: 4681669. DOI: 10.1161/CIRCGENETICS.115.001067. View

10.

Bian Z, Li L, Tang R, Hou D, Chen X, Zhang C . Identification of mouse liver mitochondria-associated miRNAs and their potential biological functions. Cell Res. 2010; 20(9):1076-8. DOI: 10.1038/cr.2010.119. View

11.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

12.

Kappler L, Li J, Haring H, Weigert C, Lehmann R, Xu G . Purity matters: A workflow for the valid high-resolution lipid profiling of mitochondria from cell culture samples. Sci Rep. 2016; 6:21107. PMC: 4759577. DOI: 10.1038/srep21107. View