Strain: C57BL/6 Gender: female Tissue: the left femurs
Extracted molecule
total RNA
Extraction protocol
The distal 5 mm of the left femurs were harvested after sacrificing by cervical dislocation. Samples were then homogenized in ISOGEN(Nippon gene, Japan) and total RNA extracted according to the manufacturer’s instructions. The extracted total RNA was cleaned up and concentrated using the RNeasy MinElute Cleanup Kit (QIAGEN, Valencia, CA) for GeneChip analysis (Proc Natl Acad Sci U S A. 2006;103(1):224-9.).
Label
Biotin
Label protocol
Total RNA was purified by using an RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. First-strand cDNA was synthesized from 5 µg of RNA by using 200 units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA), 100 pmol T7-(dT)24 primer [5'-GGCCAGTGA AT TGTA ATACGACTCACTATAGGGAGGCGG-(dT)24-3'], 1x first-strand buffer, and 0.5 mM dNTPs at 42°C for 1 h. Second-strand synthesis was performed by incubating first-strand cDNA with 10 units of Escherichia coli ligase (Invitrogen), 40 units of DNA polymerase I (Invitrogen), 2 units of RNase H (Invitrogen), 1x reaction buffer, and 0.2 mM dNTPs at 16°C for 2 h, followed by 10 units of T4 DNA polymerase (Invitrogen) and incubation for another 5 min at 16°C. Double-stranded cDNA was purified by using GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions and labeled by in vitro transcription by using a BioArray HighYield RNA transcript labeling kit (Enzo Diagnostics, Farmingdale, NY). Briefly, dsDNA was mixed with 1x HY reaction buffer, 1x biotin-labeled ribonucleotides (NTPs with Bio-UTP and BioCTP), 1x DTT, 1x RNase inhibitor mix, and 1x T7 RNA polymerase and incubated at 37°C for 4 h. Labeled cRNA was then purified by using GeneChip Sample Cleanup Module and fragmented in 1x fragmentation buffer at 94°C for 35 min.
Hybridization protocol
For hybridization to the GeneChip Mouse Expression Array 430A or 430B or Mouse Genome 430 2.0 Array (Affymetrix), 15 µg of fragmented cRNA probe was incubated with 50 pM control oligonucleotide B2, 1x eukaryotic hybridization control, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, and 1x hybridization buffer in a 45°C rotisserie oven for 16 h. Washing and staining were performed by using a GeneChip Fluidic Station (Affymetrix) according to the manufacturer's protocol.
Scan protocol
Phycoerythrin-stained arrays were scanned as digital image files with GeneChip Scanner 3000 7G with AutoLoader.
Description
4 hr estrogen treated ovariectomized wild type mouse
Data processing
Data transformation algorithm and normalization was done by Affymetrix GCOS.
Scaling:All Probe Sets, Target Signal=500
Normalization value:1
Parameters: defalut settings
