Computational protocol: PGC‐1α promotes exercise‐induced autophagy in mouse skeletal muscle

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Protocol publication

[…] Quadriceps tissue was crushed in liquid nitrogen and ~20–25 mg was weighed out for RNA isolation. Total RNA was extracted using an adapted guanidinium thiocyanate–phenol–chloroform method as previously described (Pilegaard et al. ). Pelleted RNA extracts were resuspended in 0.1 mmol/L EDTA in DEPC‐treated H2O and RNA purity and concentration was determined by spectrophotometry (Nanodrop 1000; Thermo Fisher Scientific). Reverse transcription of mRNA to cDNA was performed using Superscript II and Oligo dT (Invitrogen, Carlsbad, CA) as previously described (Pilegaard et al. ). Real‐time PCR was performed using a QuantStudio 7 Flex Real‐Time PCR System (Applied Biosystems, Waltham, MA). Primers and 5′‐FAM and 3′‐TAMRA‐labeled TaqMan probes were designed using Primer Express 3.0 software (Applied Biosystems). Primers and TaqMan probes were obtained from TAG Copenhagen (Copenhagen, Denmark). Primer and probe sequences used were: PGC‐1α FP: 5′ CTC CCT TGT ATG TGA GAT CAC GTT 3′, PGC‐1α RP: 5′ TGC GGT ATT CAT CCC TCT TGA 3′, PGC‐1α probe: 5′ ACA GCC GTA GGC CCA GGT ACG ACA 3′, LC3 FP: 5′ CGA GCT CAT CAA GAT AAT CAG AC 3′, LC3 RP: 5′ TTC CTC CTG GTG AAT GGG C 3′, LC3 probe: 5′ CGC TTG CAG CTC AAT GCT AAC CAA GC 3′. Real‐time PCR was performed in triplicates with a total reaction volume of 10 μL using Universal Mastermix II (Applied Biosystems). The cycle threshold was converted to a relative amount of target mRNA from a standard curve generated from a dilution series of a sample pool from all cDNA samples within each strain. Relative target mRNA content was normalized to total ssDNA content determined in each sample using OliGreen reagent (Molecular Probes, Leiden, The Netherlands) as previously described (Lundby et al. ). [...] Relative content of target proteins was measured in muscle lysates by SDS‐PAGE and western blotting. Equal amounts of total protein were loaded from each sample, and samples from each group and genotype were distributed on each gel. Primary antibodies were used to detect ACC2 (streptavidin–HRP; Dako, Glostrup, Denmark), phospho‐ACC Ser212 (#07‐303; Millipore, Bedford, MA), AMPKα2 (provided by Graham Hardie, Dundee University, Dundee, UK), phospho‐AMPK Thr172 (#2535), p38 (#9212), phospho‐p38 Thr180/Tyr182 (#4631), LC3A/B (#4108), p62 (#5114), phospho‐ULK1 Ser317 (#12753), phospho‐ULK1 Ser757 (#6888) (Cell Signaling, Danvers, MA). Membrane imaging and band quantification were performed using ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, UK) and ImageQuant TL v8.1 software (GE Healthcare). Protein content is expressed in arbitrary units and relative to a sample pool loaded on each side of all gels. [...] Two‐way ANOVA was used to test the effect of a single exercise bout and genotype on mRNA, proteins, phosphorylations, glycogen content, protein carbonyl content and plasma glucose. In addition, one‐way ANOVA was used to determine the effect of exercise within each genotype separately. When main effects were observed, pairwise differences were determined using Student Newman–Keuls multiple comparisons test. When equal variance test failed, values were log transformed before analysis. P < 0.05 was considered significant and 0.05 ≤ P < 0.1 was interpreted as a tendency for a significant difference. Statistical analyses were performed using Sigmaplot 12.5 software (SYSTAT, Chicago, IL). All values are presented as mean ± SE. […]

Pipeline specifications

Software tools Primer Express, Imagequant TL, SigmaPlot
Applications Miscellaneous, qPCR
Organisms Mus musculus
Diseases Muscular Diseases