|
| Status |
Public on Nov 29, 2011 |
| Title |
Mouse_BM_14M_HF diet_03 |
| Sample type |
RNA |
| |
|
| Source name |
Bone marrow
|
| Organism |
Mus musculus |
| Characteristics |
sample type: C57BL/6J Male, 14 month old
genetic background: C57BL/6J
genotype: wild type
age: 14 month old
gender: male
cell type: Bone marrow adipocyte
diet: high fat
|
| 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 obese mice Obob, male C57BL/6J mice (6-months, 14-months and 18-months of age) fed either with standard chow or high fat diet. All mice were housed in temperature-controlled conditions on a 12-h light, 12-h dark cycle.
|
| 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 as shown in Table 5.1. 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 |
the GeneChip Expression Wash, Stain and Scan User Manual (P/N702731) for the washing and staining steps required immediately after completion of hybridization. Please refer to Appendix C for fluidics protocols and fluidics script information for GeneChip ST Arrays. Scan model are Affymetrix GeneChip Fluidics Station 450 and Affymetrix GeneChip Scanner 3000 7G.
|
| Data processing |
The raw data from microarrays were analyzed using Partek Genome Suite software, version 6.3 Copyright 2008 (Partek Inc., St. Louis, MO, USA). Briefly, Affymetrix .CEL files were processed to generate gcRMA (robust multi-array average) values. This step was followed by quantile normalization and log2 transformation to represent gene expression levels.
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| |
|
| Submission date |
Feb 02, 2011 |
| Last update date |
Nov 29, 2011 |
| Contact name |
Li Fen Liu |
| E-mail(s) |
lifenliu@stanford.edu
|
| Organization name |
Stanford University
|
| Department |
Medicine
|
| Lab |
Kraemer
|
| Street address |
3801 Miranda AVE
|
| City |
Palo Alto |
| State/province |
CA |
| ZIP/Postal code |
94304 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE27017 |
Expression data of bone marrow and peripheral adipocytes from leptin deficient mice and obese mice fed with a high fat diet |
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