Computational protocol: Possible role of S-equol on bone loss via amelioration of inflammatory indices in ovariectomized mice

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Protocol publication

[…] Femora were harvested, immediately frozen in liquid nitrogen and stored at –80°C until required. Frozen bones were homogenized using a Polytron homogenizer (Kinematica, Lucerne, Switzerland). Total RNA was isolated and purified using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA target preparation for microarray analysis was performed using the GeneChip® 3’ IVT Express Kit (Affymetrix Japan K.K., Tokyo, Japan) according to the manufacturer’s instructions. Briefly, double-stranded cDNA was synthesized from the total RNA (500 ng) of each mouse with a T7-Oligo (dT) primer. After in vitro transcription to synthesize biotin-labeled aRNA, purification using Magnetic Stand-96 (Life Technologies, Tokyo, Japan) and fragmentation of the labeled aRNA were performed. Fifteen-microgram aliquots of fragmented aRNA were hybridized to an array (Mouse Genome 430 2.0 array, Affymetrix) at 45°C for 16 h. After hybridization, the gene chips were washed and stained using a GeneChip Fluidics Station 450 (Affymetrix) and then scanned (GeneChip Scanner; Affymetrix) with the GeneChip Operation Software ver. 1.4 (Affymetrix). Analysis of the DNA microarray data was performed using Microarray Suite and GeneSpring ver. 11.5 (Agilent Technologies, Santa Clara, CA). The expression level of each gene was expressed as an average of those of 5 mice in each group. The genes up- or down-regulated more than 1.5 fold in the mice femur of OVX group as compared to sham group were analyzed using Ingenuity Pathway Analysis. (Ingenuity® Systems, http://www.ingenuity.com). […]

Pipeline specifications

Software tools GeneSpring GX, IPA
Application Gene expression microarray analysis
Organisms Mus musculus, Homo sapiens
Diseases Alveolar Bone Loss, Hereditary Angioedema Type III
Chemicals Estrogens, Isoflavones