Lean and Obese Zucker Rat Extensor Digitorum Longus Muscle high-frequency electrical stimulation (HFES) Data: Regulation of MAPKs Associated Proteins.
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ABSTRACT:
SUBMITTER: Rice KM
PROVIDER: S-EPMC5723257 | biostudies-literature | 2018 Feb
REPOSITORIES: biostudies-literature
Publications
Lean and Obese Zucker Rat Extensor Digitorum Longus Muscle high-frequency electrical stimulation (HFES) Data: Regulation of MAPKs Associated Proteins.
Rice Kevin M KM Katta Anjaiah A Manne Nandini D P K NDPK Arvapalli Ravikumar R Ginjupalli Gautam K GK Wu Miaozong M Asano Shinichi S Blough Eric R ER
Data in brief 20171120
Anaerobic exercise has been advocated as a prescribed treatment for the management of diabetes: however, alterations in exercise-induced signaling remain largely unexplored in the diabetic muscle. Here, we compare the basal and the in situ contraction-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK 1/2, p38, and JNK in the lean and obese (fa/fa) Zucker rat extensor digitorum longus (EDL) muscle following a single bout of contractile stimuli. This article represents d ...[more]
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First strand cDNA probe generation from total RNA and microarray hybridization were performed according to manufacturerâs instructions (Atlas cDNA Expression Arrays User Manual, Clontech). 33P labeled cDNA was generated from sample RNA using [33P]-dATP (3000 Ci/mmol; ICN, Costa Mesa, CA) and Maloney murine leukemia virus (MMLV) reverse transcriptase (Clontech). Labeled probes were purified using nucleospin columns (Clontech). Array membranes were prehybridized in Express Hyb containing sheared Salmon testes DNA (Gibco BRL, Rockville, MD) for 30 min at 71°C in rotating hybridization tubes. Radiolabeled probe (3 x 106 cpm) was incubated in denaturing solution (Clontech) for 20 min at 71°C, after which Cot1 DNA/neutralizing solution was added and incubation continued for 10 min. The probe mixture was added to the hybridization tube and incubated with rotation overnight. Following hybridization, membranes were washed four times for 30 min in 2X saturated sodium citrate (SSC) and 1 % sodium dodecyl sulphate (SDS) at 71°C, once for 30 min in 0.1X SSC and 0.5% SDS at 71°C, and once for 5 min in 2X SSC at room temperature. Membranes were removed and immediately wrapped in plastic and exposed to a phosphor storage screen (Molecular Dynamics, Sunnyvale, CA) for four days. IV. Measurement data and specifications: Data Analysis. The membrane image was acquired using a Storm phosporimager (Molecular Dynamics) and Image Quant software. Array images were analyzed using Atlas Image 2.0 (Clontech) software to determine raw intensity levels of expression. Raw intensity data was imported into GeneSpring (Silicon Genetics, Redwood City, CA) expression analysis software. Intensity levels were normalized to the median expressed intensity on each of the arrays and averaged for the three arrays at each time point. Normalized expression levels for control arrays were established as a value of 1 and data from 6 and 72 h were expressed as fold changes relative to control. In order to simplify interpretation of the resulting dataset, a k-means cluster analysis was employed to group genes based on similarities in patterns of expression. To reduce the likelihood of false positives, the data was filtered using a criterion 2 fold change in expression prior to clustering. Miller et al. (61) have calculated that using an array of 10,000 features with a coefficient of variation (CV) of 20 percent, a 2 fold criterion for altered gene expression would result in zero false positives. The mean and median CV (15.8 and 14.3%, respectively) for all arrays in the current study fell within these guidelines for the elimination of false positives.
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