|
| Status |
Public on Oct 04, 2010 |
| Title |
muscle_exercised_SS8 |
| Sample type |
RNA |
| |
|
| Source name |
vastus lateralis, eccentrically exercised
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: vastus lateralis
gender: male
age: 21
bmi: 30.7
treatment: eccentrically exercised
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Percutaneous needle biopsies of both the exercised and non-exercised vastus lateralis muscle were obtained 3 hours following exercise. Under local anesthesia (Lidocaine), a small incision was made into the skin and fascia and the biopsy needle was inserted into the muscle. A small core of tissue (~100mg) was withdrawn and was immediately frozen in liquid nitrogen. The tissue was then stored at -80°C until it was packed and shipped in dry ice to Gene Logic (Gaithersburg, MD) for RNA extraction and expression profiling.
|
| Label |
Cy3
|
| Label protocol |
Cy3 labeled cRNA was prepared from 0.1 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%)
|
| Description |
gene expression in muscle 3h following eccentric exercise
|
| Data processing |
Probe set means from microarrays were generated from the PLIER algorithm (typically 6 iterations) in Expression Console (Affymetrix Inc., Santa Clara, CA) and imported into Partek Genomics Suite software for statistical analysis. PLIER stands for “Probe Logarithmic Intensity Error” and is a model-based signal estimator beneficial to multi-array estimations. Data was log2 transformed and then analyzed by analysis of variance (ANOVA) using age and body mass index as covariates. The data outputs were stringently filtered on the basis of p-value (p<0.005).
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| |
|
| Submission date |
Aug 18, 2010 |
| Last update date |
Sep 08, 2010 |
| Contact name |
Robert Hyldahl |
| E-mail(s) |
rhyldahl@kin.umass.edu
|
| Organization name |
University of Massachusetts Amherst
|
| Department |
Department of Kinesiology
|
| Lab |
Muscle Biology and Imaging Laboratory
|
| Street address |
Totman Building 30 Eastman Lane
|
| City |
Amherst |
| State/province |
MA |
| ZIP/Postal code |
01375 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE23697 |
Gene expression profiling in skeletal muscle 3 hours following the performance of an eccentric exercise |
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