4.6. RNA Extraction and qRT-PCR

QIAZOL Lysis Reagent and RNeasy Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany) were used for extraction of RNA [23]. RNA concentration and integrity were measured using a Nanovue Spectrophotometer (GE Healthcare) and the QIAxcel Advanced Instrument (QIAGEN), respectively. DNase I-treated total RNA (1 μg/reaction) was reverse transcribed with the QuantiTect Reverse Transcription (RT) kit (QIAGEN), using a mix of random hexamer and oligo-dT primers, as described [95]. cDNAs were amplified in duplicate by real-time PCR using Maxima SYBR Green (Thermo Scientific) or GoTaq™ qPCR Master Mix (Promega), the LightCyclerTM480 (Roche) or Rotor-GeneTMQ (QIAGEN) instruments. For transcripts encoding PGC-1α isoforms, we used primers targeting exons B1 and B4 or B5 and exon 2, to quantify the two main CNS-specific PPARGC1A transcripts. RG transcripts were quantified using primers targeting exon 1 and exon 2. Primers targeting exon 5 and exon 7A or exon 2 and the extended part of exon 3 were used to measure the transcripts encoding the class of NT-PGC-1α isoforms or a short, dominant negative isoform termed E3extended, respectively (Table S1 for primer sequences). To directly compare measurements of PPARGC1A transcripts, gene segments containing the sequences targeted by the respective transcript-specific assays were cloned and used for the construction of standard curves. The accuracy of the assays was verified by sequencing amplicons. Primers used to estimate RNA levels of genes encoding other transcripts are shown in Table S8. Relative mRNA levels were calculated using the comparative threshold cycle method (Δ_CT). Constitutively expressed RPLP0 (Ribosomal Protein, large, P0) RNA was used for normalization of mRNA abundance, as described [95]. Changes in RPLP0 transcripts resulting from activation of the CNS or RG promoters were taken into account.