Tissues were quickly removed and frozen in liquid nitrogen and stored at -80C for subsequent preparation of total RNA. Total RNA was isolated using TRI-Reagent (Molecular Research Center Inc. Cincinnati, Ohio) with modifications to remove DNA using the Qiagen RNAeasy columns and DNaseI Kit (Qiagen, Valencia, CA). RNA was stored at –70C in RNase-free H2O supplemented with the RNase inhibitor Superasin (Ambion, Austin, TX) as per manufacturers directions. Quality and quantity of RNA was determined using a Agilent Bioanalyzer as per manufacturers procedures (Agilent Technologies, Palo Alto, CA).
Label
DIG-UTP
Label protocol
1 ug of total RNA was used to transcribe DIG-labeled cRNA using Applied Biosystems Chemiluminescent RT-IVT Kit V2.0.
Hybridization protocol
Microarray hybridization (using twenty micrograms of fragmented, DIG-labeled cRNA) and processing were performed according to Applied Biosystems protocols.
Scan protocol
Chemiluminescence detection, imaging, auto gridding, and image analysis was done according to Applied Biosystems protocols and the 1700 Chemiluminescent Microarray Analyzer Software v. 1.0.3.
Description
Pooled hypothalamus RNA samples from 20 C57BL/6J mice with 'low' weight gain after 6 weeks on a high fat diet (D12331;Research Diets, New Brunswick, NJ), followed by 2 weeks of low fat diet, and 6 weeks on a high fat diet. Microarrays were performed in quadruplicate, L1, L2, L3, L4.
Data processing
Signal intensities across microarrays were normalized using the trimmed means scale (Spotfire DecisionSite for Functional Genomics, Somerville, MA).
