strain: C57BL/6 gender: Male age: 8 weeks tissue: Femur (Endosteal)
Treatment protocol
Injected with Saline (Intraperitoneal)
Extracted molecule
total RNA
Extraction protocol
Femur was removed from the mouse, cleaned with a scalpel blade and then vigorously rubbed with coarse paper towel to remove traces of muscle and periosteum. Metaphyseal zones of the bones were carefully removed using a scalpel blade. Remaining femoral diaphyses were flushed with 1 mL Phosphate Buffered Saline (PBS) plus 2% new born calf serum (NCS) (Invitrogen) using a 1mL syringe mounted with a 23-gauge needle, and reflushed up and down five times. Cells extracted into this 1mL volume of PBS were defined as the central BM fraction. These central BM cells were then pelleted via centrifugation (370g for 5 minutes) before lysis in Trizol (Invitrogen) to extract the RNA. 1mL of Trizol was flushed through each remaining empty femoral diaphysis and collected in separate tubes and defined as the endosteal RNA fraction.
Label
Biotin
Label protocol
cRNA was prepared using the Affymetrix One-Cycle Labelling Kit, using 2ug total RNA as starting material.
Hybridization protocol
10ug of fragmented biotin labelled cRNA was hybridised to a Mouse Genome 430 2.0 Array GeneChip, according to manufacturer’s instructions. GeneChip was incubated for 16hrs at 45oC in a rotating hybridisation oven at 60rpm. GeneChip was washed using Affymetrix washing script EukGE-WS2v5_450 on a Affymetrix Fluidics Station 450
Scan protocol
GeneChip was scanned on a Affymetrix GeneSchip Scanner 3000.
Data processing
The data was extracted from the CEL files using Affymetrix GeneChip Operating Software (1.4) using Affymetrix default analysis settings. Tabulated data (CHP) was generated with global scaing trimmed mean target intensity of 500.
