Quantifying Mitochondrial Transcript Levels

Steady-state levels of mitochondrial transcripts were determined by Northern blot analysis, using 3 μg of total RNA. RNA samples were separated in 1% MOPS-formaldehyde agarose gels and transferred to Hybond-N+ membranes (GE Healthcare). To analyse mitochondrial tRNA steady-state levels, samples were separated in neutral 10% PAGE and transferred to Hybond-N+ membranes (GE Healthcare). Membranes were hybridised with either randomly [³²P]-labelled dsDNA probes or in vitro transcribed single-stranded RNA probes to detect mRNAs and rRNAs or with strand-specific [³²P]-end labelled oligonucleotide probes to detect tRNAs. Membranes were exposed to a PhosphorImager screen and the signal was quantified using a Typhoon 7000 FLA and the ImageQuant TL 8.1 software (GE Healthcare). Primers used to generate dsDNA probes and oligonucleotide probes have been previously described [10 (link),23 (link),36 (link)].