strain: Wistar gender: male tissue: blood diet: high-fat diet with muberry leaf powder
Treatment protocol
Blood was collected, homogenated with TRIzol LS reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and stored at -80˚C
Growth protocol
Male Wistar rats (7 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan) and housed in a room that was maintained at 21 ± 1°C and 50 ± 10% relative humidity with a 12-h light/dark cycle (light 08:00–20:00; dark 20:00–08:00). The rats were preliminary given a normal diet (CE-2, CLEA Japan, Kanagawa, Japan) for acclimation. After a week of acclimation, the rats were divided into three groups (n=10 each) and fed for 8 weeks on a high-fat diet (Quick fat, CLEA Japan, Kanagawa, Japan), a high-fat diet plus 1% mulberry leaf powder, or a normal diet. Mulberry leaf powder was obtained from Sagamihara chamber of commerce and industry (Kanagawa, Japan). The rats were given a specific diet and water for 24 h ad libitum. On day 56 after overnight fasting, the animals were euthanized under anesthesia to collect their blood.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using TRIzol LS reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), then purified with an RNeasy mini kit (Qiagen, Valencia, CA, USA).
Label
biotin
Label protocol
Biotinylated aRNA were prepared according to the standard Affymetrix protocol from 200 ng total RNA (GeneChip® 3' IVT Express kit, Affymetrix).
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45˚C on GeneChip® Rat Genome 230 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000.
Data processing
The Affymetrix AGCC system was used to reduce the array images to the intensity values for each probe. These values were then stored in Affymatrix CEL format files and quantified with using the DFW.
