strain: CAST/EiJ tissue: femur age: sixteen-week old gender: male
Extracted molecule
total RNA
Extraction protocol
At sacrifice the diaphysis of the right femur was excised and cleaned free of soft tissue. Bone marrow was removed by flushing with PBS using a 22-guage needle and 3 ml syringe. The bone was then flash frozen in LN2 and stored at -80C. Total RNA was isolated using the Trizol Plus RNA Purification Kit (Invitrogen, Carlsbad, CA) following homogenization of the whole bone sample. RNA integrity was confirmed using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA).
Label
biotin
Label protocol
Biotin-labeled cRNA was synthesized by the total prep RNA amplification kit from Ambion (Austin, TX).
Hybridization protocol
cRNA was quantified and normalized to 77 ng/μl, and then 850 ng was hybridized to Beadchips.
Scan protocol
The hybridized Beadchips were scanned by Illumina BeadScan confocal scanner. cRNA synthesis, hybridization and scanning was performed at the Southern California Genotyping Consortium's Illumina BeadLab located at UCLA.
Data processing
The expression values were transformed using the Variance Stabilizing Transformation (VST), and normalized with the Robust Spline Normalization (RSN) algorithm using the LumiR R package. After normalization, the ComBat software was used to adjust for batch effects using an empirical Bayes method.
