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

Julian Geiger, Louise Torp Dalgaard

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Abstract

The presence of noncoding 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 qPCR following synthesis of cDNA. After qPCR-based mitomiR-profiling, the Normfinder algorithm is applied to identify the 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.
Original languageEnglish
Title of host publicationMitochondrial Bioenergetics : Methods and Protocols
EditorsCarlos M. Palmeira, António J. Moreno
Number of pages14
Place of PublicationNew York
PublisherSpringer Science+Business Media
Publication date2018
Edition2
Pages337-350
Chapter20
ISBN (Print)978-1-4939-7830-4
ISBN (Electronic)978-1-4939-7831-1
Publication statusPublished - 2018
SeriesMethods in Molecular Biology
Volume1782
ISSN1064-3745

Cite this

Geiger, J., & Dalgaard, L. T. (2018). Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells. In C. M. Palmeira, & A. J. Moreno (Eds.), Mitochondrial Bioenergetics: Methods and Protocols (2 ed., pp. 337-350). New York: Springer Science+Business Media. Methods in Molecular Biology, Vol.. 1782
Geiger, Julian ; Dalgaard, Louise Torp. / Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells. Mitochondrial Bioenergetics: Methods and Protocols. editor / Carlos M. Palmeira ; António J. Moreno. 2. ed. New York : Springer Science+Business Media, 2018. pp. 337-350 (Methods in Molecular Biology, Vol. 1782).
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Geiger, J & Dalgaard, LT 2018, Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells. in CM Palmeira & AJ Moreno (eds), Mitochondrial Bioenergetics: Methods and Protocols. 2 edn, Springer Science+Business Media, New York, Methods in Molecular Biology, vol. 1782, pp. 337-350.

Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells. / Geiger, Julian; Dalgaard, Louise Torp.

Mitochondrial Bioenergetics: Methods and Protocols. ed. / Carlos M. Palmeira; António J. Moreno. 2. ed. New York : Springer Science+Business Media, 2018. p. 337-350 (Methods in Molecular Biology, Vol. 1782).

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

TY - CHAP

T1 - Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells

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AU - Dalgaard, Louise Torp

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AB - The presence of noncoding 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 qPCR following synthesis of cDNA. After qPCR-based mitomiR-profiling, the Normfinder algorithm is applied to identify the 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.

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Geiger J, Dalgaard LT. Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells. In Palmeira CM, Moreno AJ, editors, Mitochondrial Bioenergetics: Methods and Protocols. 2 ed. New York: Springer Science+Business Media. 2018. p. 337-350. (Methods in Molecular Biology, Vol. 1782).