strain: C57BL/6J gender: Male age: 18 month old cell type: bone marrow adipocyte
Treatment protocol
Briefly, both femurs and tibias were collected after mice were sacrificed. Bones were cleaned and rinsed with 75% ethanol and DEPC water to eliminate surrounding fat and muscle cells. Fresh bone marrows were flushed out with PBS containing 1% fatty acid-free BSA and 1% RNAase and DNAase-free water using a 25-gauge needle from femurs and tibias. Red blood cells were lysed using red cell lysis buffer. After centrifugation at 3000 RPM for 5 min, floating adipocytes were collected from bone marrow stromal cells and then were washed with PBS buffer three times.
Growth protocol
Bone marrow adipocytes and peripheral white adipocytes (n=6-10 animals per group) were isolated from male C57BL/6J mice (6-months, 14-months and 18-months of age). All mice were housed in temperature-controlled conditions on a 12-h light, 12-h dark cycle, and were fed standard chow.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol (Life Technologies, Grand Island, NY, USA) and chloroform followed by purification on an RNeasy MinElute column (QIAGEN, Valencia, CA. USA). Three pooled RNA preparations were generated from 6-10 animals due to the low yield of bone marrow adipocytes during isolation.
Label
biotin
Label protocol
Whole Transcript Sense Target Labeling Assay using AmbionWT expression Kit P/N 702808.
Hybridization protocol
The GeneChip® Hybridization, Wash and Stain Kit. Affymetrix GeneChip Hybridization Oven 640 Three heating blocks are required: one at 65°C, one at 99°C, and the third one at 45°C. 1. Prepare the Hybridization Cocktail in a 1.5 mL RNase-free microfuge tube 2. Flick or gently vortex the tubes and spin down. 3. Heat the Hybridization Cocktail at 99°C for 5 minutes. Cool to 45°C for 5 minutes, and centrifuge at maximum speed for 1 minute. 4. Equilibrate the GeneChip ST Array to room temperature immediately before use. Label the array with the name of the sample that will be hybridized. 5. Inject the appropriate amount (see Table 5.2) of the specific sample into the array through one of the septa (see Figure 5.1 for location of the septa on the array). 6. Place array in 45°C hybridization oven, at 60 rpm, and incubate for 17 hours ± 1 hour. During the latter part of the array hybridization, commence preparation of the reagents required immediately after completion of hybridization.
Scan protocol
Scan model are Affymetrix GeneChip Fluidics Station 450 and Affymetrix GeneChip Scanner 3000 7G.
