Nine-week-old male, ob/ob (B6.V-Lepob/J), and ob lean control (Lepob, heterozygote from the colony) mice were purchased from Jackson Lab (Bar Harbor, Main) and acclimatized to a constant temperature and humidity (72°F and 50% relative humidity with a 12 h light and dark cycle from 05:00 h to 17:00 h) and had free access to food (LabDiet 5K20, LabDiet, St. Louis, MO) and water. At the age of twelve weeks, all animals were fasted for four hours in the morning and euthanized at 1pm under isoflurane anathesia. Nine-week-old male, C57BL/6J mice were purchased from Jackson Lab (Bar Harbor, Main) and acclimatized to a constant temperature and humidity (72°F and 50% relative humidity with a 12 h light and dark cycle from 05:00 h to 17:00 h) for one week with free access to food (LabDiet 5001, LabDiet, St. Louis, MO) and water. After acclimation, half of the mice (n=6) were kept on the regular chow diet (LabDiet 5001) and half of them (n=6) were switched to TD93075 (Harlan Teklad, Madison, Wisconsin), a high-fat diet with 54.8%Kcal from fat for four weeks. At the end of the study, all animals were fasted for four hours in the morning and euthanized at 1pm under isoflurane anesthesia.
Extracted molecule
total RNA
Extraction protocol
Gastrointestinal tissues (whole stomach and about 1cm of intestinal samples from the middle of each part of the intestine) were collected. Finally a 1cm3 segment of the inferior right lobe of the liver was taken. Segments were immediately snap frozen in liquid nitrogen and stored at -80°C for subsequent RNA extraction. RNA extraction was performed using the RNeasy Mini kit (Qiagen Inc, Valencia, Calif). Total RNA yield and purity was estimated by UV spectroscopy (Nanodrop ND-1000 Spectrophotometer; Nanodrop Technologies, Wilmington, DE, USA) and RNA quality was assessed on an Agilent 2100 Bioanalyzer. Subsequently mRNA expression analysis was carried out.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug PolyA mRNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
The biotinylated cRNA was prepared and hybridized to the Mouse 430A_2.0 GeneChip in accordance with Affymetrix protocols.
Scan protocol
GeneChips were scanned by the Affymetrix GeneChip Scanner.
Description
86213_6_ILEUM_MGI_GENE-3
Data processing
Microarray data was preprocessed using RMA implemented in R with BrainArray custom CDF.
