|
| Status |
Public on Aug 25, 2009 |
| Title |
Soleus_HS-2days_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
rat soleus muscle, hindlimb suspension 2 days
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
gender: male
age: 6 months
tissue: soleus muscle
|
| Treatment protocol |
To induce muscle atrophy, rats were hindlimb suspended for 2 or 7 days. A tail device containing a hook was attached with gauze and cynoacrylate glue while the animals were anesthetized with pentobarbital sodium (50 mg/kg body wt). After the animal regained consciousness, the tail device was connected via a thin cable to a pulley sliding on a vertically adjustable stainless steel bar running longitudinally above a high-sided cage with standard floor dimensions. The system was designed in such a way that the rats could not rest their hindlimbs against any side of the cage but could reach their food and water without difficulty.
|
| Growth protocol |
Rats were allowed free access to food and water, and were housed in a 12:12-h light-dark cycle.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from rat soleus muscle using ToTALLY RNA (Ambion, Austin, TX) according to manufacturer’s directions. RNA samples were treated with TURBO DNase to remove genomic DNA contamination and RNA integrity was assessed using the Agilent 2100 bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA); the average RIN (RNA integrity number) value for all samples was 8.4 ± 0.5 (scale 1-10) indicating high quality RNA with minimal degradation.
|
| Label |
Cy5
|
| Label protocol |
Microarray assay was performed using a service provider (LC Sciences). The assay started from 5 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (from Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry.
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| |
|
| Hybridization protocol |
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 µL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.
|
| Scan protocol |
Hybridization images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
Each chip contained 234 rat microRNA probes (Sanger miRBase 9.0) plus an additional 96 probes derived from predicted rat miRNAs. Supplementary RAW data files were provided by LC Sciences.
|
| Data processing |
Expression values were normalized by removing system related variation (sample amount variations, different labeling dyes and signal gain differences of scanners) using a locally weighted regression (cyclic LOWESS). Data adjustment included data filtering, log2 transformation, gene centering and normalization. The data filtering removed miRs with normalized intensity values below a threshold value (twice the maximum background signal) across all samples. The log2 transformation converted intensity values into log2 scale. Gene centering and normalization transformed the log2 values using the mean and the standard deviation of individual genes across all samples using the following formula: Value = [(Value) - Mean (Gene)] / [Standard deviation (Gene)]. The "A" associated with the Density measurement represents arbitrary units, and Density is a measure of the average signal intensity for each specific spot on the array.
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| |
|
| Submission date |
Aug 24, 2009 |
| Last update date |
Aug 24, 2009 |
| Contact name |
Esther E. Dupont-Versteegden |
| E-mail(s) |
eedupo2@uky.edu
|
| Phone |
(859) 323-1100
|
| Fax |
(859) 323-6003
|
| Organization name |
University of Kentucky
|
| Department |
Rehabilitation Sciences
|
| Street address |
900 S. Limestone
|
| City |
Lexington |
| State/province |
KY |
| ZIP/Postal code |
40536-0200 |
| Country |
USA |
| |
|
| Platform ID |
GPL9082 |
| Series (1) |
| GSE17776 |
MicroRNA expression in rat soleus muscle during atrophy induced by hindlimb suspension |
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