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

Julian Geiger, Louise Torp Dalgaard

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Resumé

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.
OriginalsprogEngelsk
TitelMitochondrial Bioenergetics : Methods and Protocols
RedaktørerCarlos M. Palmeira, António J. Moreno
Antal sider14
Udgivelses stedNew York
ForlagSpringer Science+Business Media
Publikationsdato2018
Udgave2
Sider337-350
Kapitel20
ISBN (Trykt)978-1-4939-7830-4
ISBN (Elektronisk)978-1-4939-7831-1
StatusUdgivet - 2018
NavnMethods in Molecular Biology
Vol/bind1782
ISSN1064-3745

Citer dette

Geiger, J., & Dalgaard, L. T. (2018). Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells. I C. M. Palmeira, & A. J. Moreno (red.), Mitochondrial Bioenergetics: Methods and Protocols (2 udg., s. 337-350). New York: Springer Science+Business Media. Methods in Molecular Biology, Bind. 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. red. / Carlos M. Palmeira ; António J. Moreno. 2. udg. New York : Springer Science+Business Media, 2018. s. 337-350 (Methods in Molecular Biology, Bind 1782).
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Geiger, J & Dalgaard, LT 2018, Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells. i CM Palmeira & AJ Moreno (red), Mitochondrial Bioenergetics: Methods and Protocols. 2 udg, Springer Science+Business Media, New York, Methods in Molecular Biology, bind 1782, s. 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. red. / Carlos M. Palmeira; António J. Moreno. 2. udg. New York : Springer Science+Business Media, 2018. s. 337-350 (Methods in Molecular Biology, Bind 1782).

Publikation: Bidrag til bog/antologi/rapportBidrag til bog/antologiForskningpeer review

TY - CHAP

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

AU - Geiger, Julian

AU - Dalgaard, Louise Torp

PY - 2018

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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. I Palmeira CM, Moreno AJ, red., Mitochondrial Bioenergetics: Methods and Protocols. 2 udg. New York: Springer Science+Business Media. 2018. s. 337-350. (Methods in Molecular Biology, Bind 1782).