CD1 wild-type (wt) mice have been placed under the following muscle wasting conditions: 1) starvation, 2) diabetes and 3) denervation. To confirm the induction of the atrophic process in each condition, we measured in EDL the expression level of Atrogin-1, a muscle specific E3-ubiquitin ligase highly induced during atrophy. 1) To induce muscle wasting by starvation, mice were food deprived for 24 and 48 hours, with free access to water. This procedure reduced body weight by 10%, gastrocnemius muscle mass by 15% and it was accompanied by more than 20 fold increase of Atrogin-1 mRNA. 2) Insulin deficiency or insulin resistance at the level of skeletal muscle tissue also leads to the activation of the atrophic program. In order to recapitulate diabetes-associated muscle atrophy, CD1 wt mice were injected intra-peritoneally with streptozotocin. This drug is recognized and approved by the “Animal Models of Diabetic Complications Consortium” and causes β-cell toxicity, resulting in primary insulin deficiency. After 7 days of an acute injection of 180 mg/Kg of Streptozotocin muscle, atrophy is already established as stated by a 4-5 fold up-regulation of atrogin-1 mRNA. 3) The absence of electrical stimulation and functional load in muscle leads to progressive atrophy. CD1 wt mice underwent sciatic denervation surgery under anaesthesia. The controlateral leg was left intact. Three different time points were analysed: 3, 7 and 14 days after surgery. Three days after surgery expression of Atrogin-1 mRNA was induce by 8–fold when compared to non-denervated muscles.
Growth protocol
Wild-type CD1 mice (Charles River) were housed in a normal environment provided with food and water. All aspects of animal care and experimentation were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and Italian regulations (DL 116/92) concerning the care and use of laboratory animals. Experimental procedures were approved by the local Ethical Committee of the University of Padova.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from muscle biopsies by using Trizol® Reagent (Life Technologies, CA) according to the manufacturer’s protocol. The commercially available PureLink™ miRNA Isolation Kit (Life Technologies) was used to enrich total RNA preparation for small RNA molecules (< 200 bp). Total RNA was quantified using the ND-1000 spectrophotometer (Nanodrop, Wilmington, DE), while RNA integrity and content of microRNAs (%) in each samples were assessed by capillary electrophoresis using the RNA 6000 Nano LabChip and the Small RNA Nano LabChip respectively using the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Only total RNA samples with R.I.N. (RNA Integrity Number) values of 6, or higher, and the percentage of miRNA < 20% were used for microarray analysis.
Label
Cy5
Label protocol
Small RNA molecules (< 200 nt) were labelled with the mirVana Labeling Kit and amine-reactive dyes as recommended by the manufacturer (Ambion). Poly(A) polymerase and a mixture of unmodified and amine-modified nucleotides were used first to append a poly-nucleotide tail to the 3’ end of each miRNA. The amine-modified miRNAs were then cleaned up and coupled to NHS-ester modified Cy5 or Cy3 dyes (GE Healthcare). Unincorpored dyes were removed with a second glass fiber-based cleanup procedure as described by Shingara et al. (2005).
CD1 wild-type (wt) mice have been placed under the following muscle wasting conditions: 1) starvation, 2) diabetes and 3) denervation. To confirm the induction of the atrophic process in each condition, we measured in EDL the expression level of Atrogin-1, a muscle specific E3-ubiquitin ligase highly induced during atrophy. 1) To induce muscle wasting by starvation, mice were food deprived for 24 and 48 hours, with free access to water. This procedure reduced body weight by 10%, gastrocnemius muscle mass by 15% and it was accompanied by more than 20 fold increase of Atrogin-1 mRNA. 2) Insulin deficiency or insulin resistance at the level of skeletal muscle tissue also leads to the activation of the atrophic program. In order to recapitulate diabetes-associated muscle atrophy, CD1 wt mice were injected intra-peritoneally with streptozotocin. This drug is recognized and approved by the “Animal Models of Diabetic Complications Consortium” and causes β-cell toxicity, resulting in primary insulin deficiency. After 7 days of an acute injection of 180 mg/Kg of Streptozotocin muscle, atrophy is already established as stated by a 4-5 fold up-regulation of atrogin-1 mRNA. 3) The absence of electrical stimulation and functional load in muscle leads to progressive atrophy. CD1 wt mice underwent sciatic denervation surgery under anaesthesia. The controlateral leg was left intact. Three different time points were analysed: 3, 7 and 14 days after surgery. Three days after surgery expression of Atrogin-1 mRNA was induce by 8–fold when compared to non-denervated muscles.
Growth protocol
Wild-type CD1 mice (Charles River) were housed in a normal environment provided with food and water. All aspects of animal care and experimentation were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and Italian regulations (DL 116/92) concerning the care and use of laboratory animals. Experimental procedures were approved by the local Ethical Committee of the University of Padova.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from muscle biopsies by using Trizol® Reagent (Life Technologies, CA) according to the manufacturer’s protocol. The commercially available PureLink™ miRNA Isolation Kit (Life Technologies) was used to enrich total RNA preparation for small RNA molecules (< 200 bp). Total RNA was quantified using the ND-1000 spectrophotometer (Nanodrop, Wilmington, DE), while RNA integrity and content of microRNAs (%) in each samples were assessed by capillary electrophoresis using the RNA 6000 Nano LabChip and the Small RNA Nano LabChip respectively using the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Only total RNA samples with R.I.N. (RNA Integrity Number) values of 6, or higher, and the percentage of miRNA < 20% were used for microarray analysis.
Label
Cy3
Label protocol
Small RNA molecules (< 200 nt) were labelled with the mirVana Labeling Kit and amine-reactive dyes as recommended by the manufacturer (Ambion). Poly(A) polymerase and a mixture of unmodified and amine-modified nucleotides were used first to append a poly-nucleotide tail to the 3’ end of each miRNA. The amine-modified miRNAs were then cleaned up and coupled to NHS-ester modified Cy5 or Cy3 dyes (GE Healthcare). Unincorpored dyes were removed with a second glass fiber-based cleanup procedure as described by Shingara et al. (2005).
Hybridization protocol
Microarray hybridization was carried out in a dual slide chamber (HybChamber, Gene Machines, San Carlos, CA, USA) humidified with 100 μL of 3×SSC. Labeled RNA was dissolved in 6 μL of 3X miRNA Hybridization Buffer (Ambion), denatured at 95°C for 3 min and applied directly to the slides. Microarrays were covered with a 24×24 mm cover slip and hybridized for 21 hours at 42°C by immersion in a high precision water bath (W28, Grant, Cambridge, UK). Hybridized slides were successively washed in: Low and High Stringency Buffer (Ambion) for 30 sec. and dried by centrifugation (500 x g).
Scan protocol
Array scanning was carried out using a GSI Lumonics LITE dual confocal laser scanner with a ScanArray Microarray Analysis System (Perkin Elmer), and raw images were analyzed with QuantArray Analysis Software (GSI Lumonics, Ottawa, Canada).
Description
miRNA expression profiling of the gastrocnemius muscle from CD1 denervated-mice (14 days, 699-B)
Data processing
Inter-array normalization of expression levels was performed with loessM+GPA (Risso et al., 2009 - Bioinformatics, 25:2685-91). Principal component analysis, cluster analysis and profile similarity searching were performed with tMev that is part of the TM4 Microarray Software Suite (Saeed et al., 2006 - Methods in Enzymology, 411:134-93). In particular, hierarchical cluster analysis was performed with Pearson correlation or Euclidean distance coefficient as distance measure with complete linkage. Identification of differentially expressed miRNAs was performed with ANOVA one class analysis (p-value <0.05) (Pan et al., 2002 - Bioinformatics, 18: 546-5).
