Gcs 3000 7g scanner

The DIO, lean, and db/db mice were injected intraperitoneally with vehicle or celastrol one time (250 μg/kg) and sacrificed shortly thereafter 6 hours, or injected once per day (100 μg/kg) for one day, or four days, and sacrificed 6 hours after the final injection. The hypothalami were extracted and stored at −80 °C until RNA extraction. To extract total RNA, 500 μl of TRIzol was added to each sample. The tissues were homogenized with a bench-top TissueLyser II (Qiagen, Valencia, CA), and the hypothalamic RNA was extracted according to the manufacturer’s instruction of TRIzol lysis reagent. For microarray analysis, the extracted total RNA was cleaned using RNeasy Min Cleanup Kit (74104, QIAGEN) and 1 μg total RNA was used for microarray analysis in Molecular Biology Core Facilities of Dana Farber Cancer Institute-Harvard Medical School. Briefly, the total RNA was processed using the Affymetrix GeneChip™ WT Reagent Kit. The kit generates amplified and biotinylated sense-stranded DNA targets for hybridization. Fragmented, biotinylated cDNA was hybridized to Mouse Gene 1.0 ST arrays for 16 h at 45 °C and 60 rpm in an Affymetrix GeneChip™ Hybridization Oven 645. Mouse GeneChip ST arrays were washed and stained using the Affymetrix FS450 automated fluidics station. GeneChips were scanned in an Affymetrix GCS3000 7G scanner with autoloader.