Strain: ICR Gender: Male Age: 6 months Body weight: 28.67g Tissue: Bone
Biomaterial provider
Samples were collected by Melissa A. Smith, Department of Physiology, University of Kentucky, 800 Rose Street, Lexington, Kentucky 40536-0298, USA
Treatment protocol
Hindlimb muscles were unloaded using methods described previously (Matuszczak, Arbogast, Reid 2004). Briefly, Elastoplast tape (Beiersdorf-Jobst, Rutherford College, NC) was used to wrap the tail of each animal. A metal clip on the tape was attached to a nylon monofilament line via a stainless steel swivel. The distal end of the nylon line was attached to an overhead support and shortened to suspend the animal in a 45° head-down tilt position. The swivel enabled the animal to explore the cage (360° range of motion) and obtain food and water freely. Animals were observed daily for changes in appearance and activity. Each animal was weighed and the angle of hindlimb suspension was adjusted if necessary. After 24hr, each animal was deeply anesthetized in the hindlimb-unloaded condition. The animal was removed from the unloading device, the femur bones were excised with the animal under surgical anesthesia, and the animal was euthanized. Bone was immediately placed in RNAlater (Ambion, Foster City, CA) and stored until RNA extraction.
Growth protocol
Mice were maintained on a reversed 12:12-h light-dark cycle with controlled temperature (21 ± 1°C) and humidity. Food and water were provided ad libitum. At 1 wk after arrival, the animals were separated into individual cages for a 3-day acclimation period then were randomly assigned to either of two experimental groups: freely ambulating controls and hindlimb-unloaded animals.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the Qiagen RNeasy kit Qiagen. The integrity of the RNA was confirmed with analysis by the Agilent 2100 bioanalyser using the RNA 6000 LabChip kit.
Label
Cy3
Label protocol
600 ng RNA and 3 µl Agilent One-Color RNA Spike-In RNA were labelled with the Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color according to Manufacture’s instructions as follows: 1.2 µl T7 Promoter Primer was added to 600 ng RNA and 3 µl spike in control and denatured at 65 ºC. First Strand Buffer, DTT, dNTP MMLV and RNaseOut was added. The cDNA was synthesised during the following incubation step (2h at 40 ºC). After 10 min denaturation at 65 ºC and the addition of Cy-labelled CTP, Transcription Buffer, DTT, NTP, PEG, RNaseOUT, Inorganic Phosphatase, and T7 RNA Polymerase the synthesis of the fluorescent labelled cRNA was performed during the second incubation step (2 h at 40 ºC). The labelled cRNA was purified with the Qiagen RNeasy Mini Kit according to Manufacturer’s protocol.
Hybridization protocol
The Agilent Hybridisation Kit (catalogue number 5188-5242) was used in conjunction with Agilent Mouse Oligo Arrays (catalogue number G4122F). 2μg of the labelled sample RNA were used for hybridisation according to the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol The hybridisation was performed for 17 h at 65 ºC at 10 rpm. Slides were them washed for 1 min at 22 ºC in Wash Solution 1 (catalogue number 5188-5325) and 1 min at 22 ºC in Wash Solution 2, pre-warmed to 37 ºC (catalogue number 5188-5326). Slides were incubated for 30s in Agilent Stabilisation and Drying Solution (catalogue number 5185-5979).
Scan protocol
Microarrays were scanned using the Agilent Technologies Scanner G2505B US45102871 (ChipScan software version A.7.0.1), scan region 61 X 21.6mm, scan resolution 5 micron single pass, extended dynamic range selected (XDR Hi 100%; XDR Lo 10%).
Description
NA
Data processing
Data was extracted from the scanned image using the Agilent Feature Extraction Software version. 9.1.3.1 (protocol GE1-v5_91_0806). The VALUE column came from the gProcessedSignal column of the result table.
