Physiological functions of the RORs in brown adipose tissue

To determine the physiological targets of the RORs in brown adipose tissue. Keywords: Nuclear receptors, RORs, adipose Gene expression analysis was conducted using Agilent whole mouse genome arrays (012694). The analysis was perfomed in duplicate, employing a fluor reversal. RNA was isolated from interscapular brown adipose tissue from C57BL/6 male WT or DKO (RORa-/- and RORg-/-) 8-12 week old mice around circadian time 20. Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturer’s protocol. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours 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 were retrieved with the Agilent Feature Extraction software (v7.5), using defaults for all parameters. The Agilent Feature Extraction Software performed error modeling, adjusting for additive and multiplicative noise. The resulting data were processed using the Rosetta Resolver® system (Rosetta Biosoftware, Kirkland, WA).