Affymetrix GeneChip Murine Genome U74Av2 [MG_U74Av2]

Effects of diet and genetic background on sterol regulatory element-binding protein-1c, stearoyl-CoA desaturase 1, and the development of the metabolic syndrome.

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Both environmental and genetic factors play important roles in the development of the metabolic syndrome. To elucidate how these factors interact under normal conditions, C57Bl/6 (B6) and 129S6/SvEvTac (129) mice were placed on a low-fat or high-fat diet.  Liver samples were extracted and hybridized to Affymetrix Genome U74 (version 2) arrays.

B6 vs. 129

High-Fat vs. Low-Fat

Transcription profiling of liver from B6 and 129 mice fed on low and high fat diets

Both environmental and genetic factors play important roles in the development of the metabolic syndrome. To elucidate how these factors interact under normal conditions, C57Bl/6 (B6) and 129S6/SvEvTac (129) mice were placed on a low-fat or high-fat diet.  Liver samples were extracted and hybridized to Affymetrix Genome U74 (version 2) arrays.


cbil.upenn.edu:RAD.Protocol:263:Protocol

The fragmented cRNA is mixed with the hybridization cocktail, and heat shocked at 99C for 5 minutes.  In the meanwhile, the probe array is filled with hybridization buffer and incubated at 45C for 10 minutes with rotation.  The heated hybridization cocktail is then transferred to a 45C heat block for 5 minutes, and spun at maximum speed in a microcentrifuge for 5 minutes.  Fill the probe array cartridge with the clarified hybridization cocktail and place probe array in rotisserie box in 45C oven. Hybridize for 16 hours.

Affymetrix MAS 5.0 Probe Cell Analysis

The Cell Analysis algorithm calculates a single intensity value for each probe cell on an array. This intensity value, representative of the hybridization level of its target, is derived as follows. The bordering pixels of the probe cell are excluded. The remaining pixel intensity distribution is calculated, and the intensity value associated with 75% of the distribution is used as the probe cell intensity. The intensities for all probe cells are saved in the .CEL file.

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An absolute expression analysis examines the cell intensity file (*.CEL) from one experiment. For each transcript represented on the probe array, the expression algorithm computes the 1) detection call [present, absent, or marginal (unable to call the transcript present or absent), or no call]; 2) detection p-value; 3) signal (background subtracted and adjusted for noise); 4) stat pairs; 5) stat pairs used. The expression values are saved in the .CHP file.

cbil.upenn.edu:RAD.Protocol:3547:Protocol

The RNeasy procedure represents a novel technology for RNA isolation. This technology combines the selective binding properties of a silica-gel-based membrane with the speed of microspin technology. A specialized high-salt buffer system allows RNA to bind to the RNeasy silica-gel membrane. Biological samples are first lysed and homogenized in the presence of a highly denaturing guanidine isothiocyanate (GITC)-containing buffer, which immediately inactivates RNases to ensure isolation of intact RNA. Ethanol is added to provide appropriate binding conditions, and the sample is then applied to an RNeasy column where the total RNA binds to the membrane and contaminants are efficiently washed away. High-quality RNA is then eluted in water.

cbil.upenn.edu:RAD.Protocol:4731:Protocol

The LFD derives 14% calories from fat, 25% calories from protein, and 61% calories from carbohydrates and was found to contain 1.5% saturated fatty acids, 2.7% monounsaturated fatty acids (MUFAs), and 0.6% polyunsaturated fatty acids by weight.  Arachidonic acid was undetectable. The mice were maintained on a 12-h light-dark cycle; unless otherwise indicated, serum samples were taken and mice were killed between 9:00 and 11:00 A.M., in the nonfasted state at 6 months of age. Insulin levels were measured in plasma samples of random fed mice using the Crystal Chem ELISA kit and mouse insulin standards. Three independent cohorts were used to perform these experiments.

cbil.upenn.edu:RAD.Protocol:4732:Protocol

The HFD derives 55% calories from fat, 21% calories protein, and 24% calories from carbohydrates and was found to contain 4.2% saturated fatty acids, 5.0% MUFAs, and 11.2% polyunsaturated fatty acids. The LFD and HFD have 15.4 and 7.1 mg cholesterol per 100 g, respectively. Arachidonic acid was undetectable. The mice were maintained on a 12-h light-dark cycle; unless otherwise indicated, serum samples were taken and mice were killed between 9:00 and 11:00 A.M., in the nonfasted state at 6 months of age. Insulin levels were measured in plasma samples of random fed mice using the Crystal Chem ELISA kit and mouse insulin standards. Three independent cohorts were used to perform these experiments.

cbil.upenn.edu:RAD.Protocol:925:Protocol

See pages 2.1.10-2.1.17 of the GeneChip Expression Analysis Technical Manual for all details. First-strand cDNA is synthesized from (5.0 to 20.0 ug) high-quality total RNA, using the GeneChip T7-Oligo(dT) Promoter Primer Kit or random primers provided with the SuperScript Choice Kit. Second-strand synthesis is carried out in: 1X second-strand reaction buffer, 200 uM each dNTP mix, 10 U E. coli DNA ligase, 40 U E. coli DNA polymerase I, 2 U E. coli RNase H in a final volume of 150 uL. The double-stranded cDNA is cleaned-up using the components supplied with the GeneChip Sample Cleanup Module. Enzo BioArray HighYield RNA Transcript Labeling Kit (Affymetrix, P/N 900182) is used to synthesize biotin-labeled cRNA target. The latter is cleaned up using the components supplied with the GeneChip Sample Cleanup Module.

Transcription profiling of liver from B6 and 129 mice fed on low and high fat diets

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