dsRNA after immunoprecipitation was purified by TRI Reagent (Sigma) using the manufacturer’s protocol. 20% of dsRNA eluate was dissolved in denaturing solution and run on a 1% agarose gel as described previously11 (link). Subsequently, RNA was transferred to Amersham Hybond-N+ membrane (GE Healthcare Life Sciences) and UV cross-linked. For detection of mitochondrial transcripts probes were labelled with [α-32P] dATP (Hartmann Analytic) using a DECAprime II Kit (Ambion). PCR products corresponding to the following fragments of human mtDNA were used as templates: 254–4469 (Probe 1), 4470–8365 (Probe 2), 8365–12137 (Probe 3), 12091–16024 (Probe 4). Hybridizations were performed in PerfectHyb Plus buffer (Sigma) at 65 °C. Membranes were exposed to PhosphorImager screens (FujiFilm), which were scanned following exposure by a Typhoon FLA 9000 scanner (GE Healthcare). Data were analysed by Multi Gauge v.3.0 software (FujiFilm).
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Quantification of Mitochondrial Transcripts
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Corresponding Organization : University of Oxford
Other organizations : Institute of Biochemistry and Biophysics, Polish Academy of Sciences, University of California, Los Angeles, Institut des Maladies Génétiques Imagine, Inserm, University of Manchester, Institut Pasteur, MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Warsaw
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Variable analysis
independent variables
- Immunoprecipitation of dsRNA
- Purification of dsRNA using TRI Reagent
- Denaturing and running dsRNA on a 1% agarose gel
- Transferring RNA to Amersham Hybond-N+ membrane and UV cross-linking
- Labeling of probes with [α-32P] dATP using a DECAprime II Kit
- Detection of mitochondrial transcripts on the membrane
- Manufacturer's protocol for TRI Reagent purification
- Previously described method for denaturing and running dsRNA on a 1% agarose gel (ref. 11)
- Hybridization conditions (PerfectHyb Plus buffer at 65 °C)
- PCR products corresponding to the following fragments of human mtDNA: 254–4469 (Probe 1), 4470–8365 (Probe 2), 8365–12137 (Probe 3), 12091–16024 (Probe 4)
- Not explicitly mentioned
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