Normal and X-linked hypophosphatemic, Hyp, mice were bred in our colony on the C57BL/6J background from stock originating from the Jackson Laboratory, Bar Harbor, ME. The breeders were fed Teklad Mouse Breeder Diet (W) No. 8626 (Harlan, Madison, WI) and tap water ad lib. Pups were weaned at three weeks of age and fed Teklad Rodent Diet (W) No. 8604 (Harlan). At five weeks of age, Hyp mice (hemizygous male (Hyp/Y) and heterozygous female (Hyp/+)) and littermate normal (wild type) male and female mice were euthanized, and both femora were harvested. The femora were cut at the metaphyseal/diaphyseal junction and the diaphyses were flushed with physiological saline to remove as much marrow as possible. The resulting shafts were frozen in liquid nitrogen and stored at 70 C.
Extracted molecule
total RNA
Extraction protocol
Samples were processed as described in the Affymetrix GeneChip Expression Analysis Technical Manual (copyright 2004, Affymetrix, Inc., Santa Clara, CA, Rev. 6, Part number 701025, http://www.affymetrix.com). The sample preparation is described here in brief. The femora were weighed and homogenized, and total RNA was extracted with TRIzol (Invitrogen Life Technologies, Carlsbad, CA).
Label
Biotin-labeled cRNA
Label protocol
Samples were processed as described in the Affymetrix GeneChip Expression Analysis Technical Manual (copyright 2004, Affymetrix, Inc., Santa Clara, CA, Rev. 6, Part number 701025, http://www.affymetrix.com). Samples of RNA were purified on RNeasy columns from Qiagen (Valencia, CA, P/N 74104) and then 8 μg purified RNA were converted to double-stranded cDNA with an Affymetrix One-Cycle cDNA Synthesis Kit (P/N 900431). The cDNA was then expressed as biotin-labeled cRNA by in vitro transcription (IVT) with the 3’ Amplification Reagents for IVT Labeling (Affymetrix P/N 900449). Each sample was spiked with bioB, bioC, bioD, and cre (Affymetrix P/N 900299). The biotin-labeled cRNA was fragmented non-enzymatically.
Hybridization protocol
Samples were processed as described in the Affymetrix GeneChip Expression Analysis Technical Manual (copyright 2004, Affymetrix, Inc., Santa Clara, CA, Rev. 6, Part number 701025, http://www.affymetrix.com). The fragmented cRNA from the 20 independent samples was hybridized to 20 mouse 430 2.0 GeneChip microarrays (Affymetrix P/N 520029) in Affymetrix hybridization buffer for 16 hours at 45 C. The hybridized arrays were washed and stained in the Affymetrix Fluidics Station 400 to attach fluorescent labels to the biotin, followed by biotin-labeled antibody, and then a second staining with fluorescent labeling of the biotin.
Scan protocol
Each array was scanned once with the Affymetrix GeneArray Scanner 3000.
Description
Experimental design. Equal amounts of RNA from two mice, matched for genotype and sex were pooled to create each sample for microarray analysis. Four treatment groups were done: (1) Normal male mice, (2) Hyp male mice, (3) normal female mice, and (4) Hyp female mice. Five replicates were done with each replicate containing one sample from each of the four treatment groups for a total of 20 independent samples (40 mice total). Each replicate was matched for littermates and parallel processing.
Data processing
The data were analyzed with Affymetrix GeneChip Operating System (GCOS) 1.2. GCOS was used for quality control for each array and to scale the mRNA expression (signal value) of each gene so that the average of all genes is set to 500. GCOS was used to create a single pivot table with 45,101 genes for all 20 samples.
