tissue: gastrocnemius muscle treatment: control strain: CD1
Treatment protocol
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. Total RNA, enriched for long RNA molecules (> 200 nt) by using PureLink™ miRNA Isolation Kit (Life Technologies), was quantified using the ND-1000 spectrophotometer (Nanodrop, Wilmington, DE), while RNA integrity in each samples were assessed by capillary electrophoresis using the RNA 6000 Nano LabChip using the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Only total RNA samples with R.I.N. (RNA Integrity Number) values of 6 were used for microarray analysis.
Label
Cy5
Label protocol
Total RNA (> 200 nt) samples were labeled using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion), in accordance with the manufacturer's instructions. First strand synthesis with an engineered reverse transcriptase should produce virtually full-length cDNA, which is the best way to ensure reproducible microarray results. The use of a modified oligo(dT) primer bearing a T7 promoter (Van Gelder et al., 1990) allows the next amplification steps: after second strand synthesis and clean-up the cDNA becomes a template for in vitro transcription with T7 RNA polymerase. About 5 µg aminoallyl-labeled aRNA were coupled with Cy5 or Cy3 dyes (GE Healthcare) and purified on column (Ambion).
tissue: gastrocnemius muscle treatment: denervated time: 14 days strain: CD1
Treatment protocol
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. Total RNA, enriched for long RNA molecules (> 200 nt) by using PureLink™ miRNA Isolation Kit (Life Technologies), was quantified using the ND-1000 spectrophotometer (Nanodrop, Wilmington, DE), while RNA integrity in each samples were assessed by capillary electrophoresis using the RNA 6000 Nano LabChip using the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Only total RNA samples with R.I.N. (RNA Integrity Number) values of 6 were used for microarray analysis.
Label
Cy3
Label protocol
Total RNA (> 200 nt) samples were labeled using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion), in accordance with the manufacturer's instructions. First strand synthesis with an engineered reverse transcriptase should produce virtually full-length cDNA, which is the best way to ensure reproducible microarray results. The use of a modified oligo(dT) primer bearing a T7 promoter (Van Gelder et al., 1990) allows the next amplification steps: after second strand synthesis and clean-up the cDNA becomes a template for in vitro transcription with T7 RNA polymerase. About 5 µg aminoallyl-labeled aRNA were coupled with Cy5 or Cy3 dyes (GE Healthcare) and purified on column (Ambion).
Hybridization protocol
Labeled targets (atrophic and control muscle) were mixed and ethanol precipitated. After dissolving the pellet in 120 µl of hybridization buffer (5X SSC, 0.1% SDS, 25% formamide), samples were denatured at 90°C for 2 min and added to the microarrays. Prehybridization was performed overnight at 48°C in the presence of 5X SSC, 5X Denhardt, 0.1% SDS, 100 ng/µl ss-DNA. Competitive hybridizations were carried on for 48 hours at 46°C in an ArrayBooster microarray incubator (Advalytix), followed by a series of post-hybridization washings (Chemello et al., 2011).
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
Gene expression profiling of the gastrocnemius muscle from CD1 denervated-mice (14 days, 698)
Data processing
Normalization of the expression levels was performed with MIDAW tool (Romualdi et al., 2005 - Nucleic Acids Res. 33: W644-9). Principal component analysis, cluster analysis and profile similarity searching were performed with tMev that is part of the TM4 Microarray Software Suite (Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, et al. Methods in Enzymology, 2006;411:134-93). Identification of differentially expressed genes was performed with one and two class Significance Analysis of Microarray (SAM) program (Tusher VG, Tibshirani R, Chu G. Proc Natl Acad Sci USA 2001;98:5116-121) with default settings. SAM uses a permutation-based multiple testing algorithm and identifies significant genes with variable false-discovery rates (FDR). This can be manually adjusted to include a reasonable number of candidate genes with acceptable and well defined error probabilities
Differentially expressed genes involved in the regulation of muscle wasting during catabolic conditions
Data table header descriptions
ID_REF
VALUE
average Log(2) ratio of the two replicates intensities normalized by global mean normalization by MIDAW software (Romualdi et al. 2005) , defined as Channel 2 divided by Channel 1 (atrophic model/control) (data used in our work)
