Nytran n

Northern hybridization was performed essentially as described previously [139 (link)]. 2.5 μg of mitochondrial RNA was separated on a 1 % denaturing formaldehyde gel, transferred onto Nytran N nylon membrane (GE Healthcare) and hybridized with the appropriate probe. Fragments of respective mitochondrial genes amplified by PCR and cloned into plasmid vectors were radiolabeled with α-³²P-dATP using the Nick Translation System (Invitrogen). For the detection of ATP6, ATP8, ATP9 oligonucleotide probes labeled with γ-³²P-ATP using the T4 polynucleotide kinase (New England Biolabs) were used. All the probes used in Northern blot analysis are listed in Additional file 5: Table S5.

Southern blot analysis was performed under high stringency conditions as described previously (Feliciello et al. 2011 (link)). Primers 2ab were used to prepare a biotinylated probe using as a template purified Tcast2a (360 bp) or Tcast2b (179 bp) repeat amplified by PCR from genomic DNA. Results obtained with the two types of probes were equivalent. For quantitative dot-blot analysis DNA samples at 1 mg/ml in denaturing buffer (0.4M NaOH, 1M NaCl) were serially diluted with an equal volume of herring sperm DNA (Promega) at the same concentration of 1 mg/ml in denaturing buffer. A 200 µl aliquot of each dilution was added per slot in a dot-blot apparatus (Bio-Rad), filtered on a nylon membrane Nytran N (Amersham Hybond—GE Healthcare) and fixed on a membrane by baking at 80 °C. As a standard, a purified sample of a specific PCR-amplified Tcast2a and Tcast2b from genomic DNA was used at an initial concentration of 3 ng. Hybridization and staining procedures were the same as those used for Southern blot analysis. Biotinylated probe is a Tcast2a specific subunit obtained using primers P1 and 2aRev.