For microarray analysis, total RNA was isolated from adductor muscles using the RNeasy Fibrous Tissue Minikit (QIAGEN). RNA concentration, purity, and integrity were analyzed by nanodrop (nanodrop Technologies) and bioanalyzer (Agilent 2000) measurements.
For microRNA expression profiling, adductor muscle tissue from days 0, 1, 3, and 7 after induction of hindlimb ischemia was used. MicroRNA expression profiling was performed as described previously using locked nucleic acid (LNA)-based arrays (miRCURY LNA miR Array Ready-to-Spot Probe Set, Exiqon).27 (link) Normalization and background correction were performed in the “statistical language R” using the “vsn” package (Bioconductor). Differential expression was assayed using the “limma” package (Bioconductor) by fitting the eBayes linear model and contrasting individual treatments with untreated controls. Log2 fold changes were calculated using the toptable function of the limma package.4 (link), 45 (link)
For whole-genome expression profiling, adductor muscle tissue from days 0, 1, 3, 7, 14, and 28 after induction of hindlimb ischemia was used. Whole-genome expression profiling was performed using MouseWG-6 v2.0 Expression Beadchips (Illumina), and expression levels were Log2-transformed as described previously.27 (link), 43 (link)
For microRNA expression profiling, adductor muscle tissue from days 0, 1, 3, and 7 after induction of hindlimb ischemia was used. MicroRNA expression profiling was performed as described previously using locked nucleic acid (LNA)-based arrays (miRCURY LNA miR Array Ready-to-Spot Probe Set, Exiqon).27 (link) Normalization and background correction were performed in the “statistical language R” using the “vsn” package (Bioconductor). Differential expression was assayed using the “limma” package (Bioconductor) by fitting the eBayes linear model and contrasting individual treatments with untreated controls. Log2 fold changes were calculated using the toptable function of the limma package.4 (link), 45 (link)
For whole-genome expression profiling, adductor muscle tissue from days 0, 1, 3, 7, 14, and 28 after induction of hindlimb ischemia was used. Whole-genome expression profiling was performed using MouseWG-6 v2.0 Expression Beadchips (Illumina), and expression levels were Log2-transformed as described previously.27 (link), 43 (link)
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