Analysis and validation of a new extended method for estimating plasma free cortisol including neutrophil elastase and competition from other steroids

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

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
TidsskriftThe Journal of Steroid Biochemistry and Molecular Biology
Vol/bind181
Sider (fra-til)109-124
Antal sider16
ISSN0960-0760
DOI
StatusUdgivet - jul. 2018

Emneord

  • Cortisol
  • Corticosteroid-binding globulin
  • Mechanism based model
  • Progesterone
  • Testosterone
  • Neutrophil elastase

Citer dette

@article{c7aec33c25de40a99553586c4a0f60fe,
title = "Analysis and validation of a new extended method for estimating plasma free cortisol including neutrophil elastase and competition from other steroids",
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.",
keywords = "Cortisol, Corticosteroid-binding globulin, Mechanism based model, Progesterone, Testosterone, Neutrophil elastase",
author = "Johanne Gudmand-H{\o}yer and Ottesen, {Johnny T.}",
year = "2018",
month = "7",
doi = "10.1016/j.jsbmb.2018.04.003",
language = "English",
volume = "181",
pages = "109--124",
journal = "The Journal of Steroid Biochemistry and Molecular Biology",
issn = "0960-0760",
publisher = "Pergamon Press",

}

TY - JOUR

T1 - Analysis and validation of a new extended method for estimating plasma free cortisol including neutrophil elastase and competition from other steroids

AU - Gudmand-Høyer, Johanne

AU - Ottesen, Johnny T.

PY - 2018/7

Y1 - 2018/7

N2 - 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.

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.

KW - Cortisol

KW - Corticosteroid-binding globulin

KW - Mechanism based model

KW - Progesterone

KW - Testosterone

KW - Neutrophil elastase

U2 - 10.1016/j.jsbmb.2018.04.003

DO - 10.1016/j.jsbmb.2018.04.003

M3 - Journal article

VL - 181

SP - 109

EP - 124

JO - The Journal of Steroid Biochemistry and Molecular Biology

JF - The Journal of Steroid Biochemistry and Molecular Biology

SN - 0960-0760

ER -