Identification of genes regulated by RORg in mouse thymus

Microarray analysis was performed in order to identify changes in gene expression in thymocytes isolated from RORg-/- mice in comparison to those of wild type mice. Keywords: Genetic Modification Microarray analyses were carried out by the NIEHS Microarray Group (NMG). Gene expression analysis was conducted using Agilent Mouse arrays (Agilent Technologies, Palo Alto, CA) representing about 20,000 genes. For each condition, equal amounts of total RNA from thymocytes of three individual wild type or RORg-/- mice were pooled and amplified. Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 1 mg of total RNA, Cy3- or Cy5-labeled cRNA was synthesized according to manufacturer’s protocol. For each comparison, 750 ng of each Cy3- and Cy5-labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 h in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data was obtained with the Agilent Feature Extraction software (v7.1) using defaults for all parameters.