hGRβ: Elucidation of its Role in Transcriptional Regulation and Identification of a Ligand. Homo sapiens

The human glucocorticoid receptor (GR) is expressed as two alternately spliced Cterminal isoforms: α and β. In contrast to the canonical hGRα, hGRβ is constitutively nuclear, is not thought to bind ligand, and is believed to affect gene transcription only by acting as a dominant negative to hGRα. Microarray analysis indicated that hGRβ, expressed in the absence of hGRα, can regulate gene expression, and furthermore, occupation of hGRβ with the antagonist RU-486 diminishes that capacity. Keywords: cell type comparison Overall design: RNA preparation: Total RNA was extracted from 5x10 6 U-OFF or U-2 OSβ cells treated with either ethanol vehicle or 1μM RU-486 for 6 hours using the RNAqueous Total RNA Isolation Kit (Ambion Inc. Austin, TX) according to manufacturer’s instructions. RNA was treated with DNase using the DNA-Free DNase Treatment and Removal Reagents (Ambion Inc.) according to manufacturer’s instructions prior to use with the microarray. Four pairs of RNA (vehicle vs. RU-486 treated) were harvested for each cell type to yield four biological replicates for gene expression analysis. Linear Amplification Label Protocol and Feature Extraction: Gene expression analysis was conducted using Agilent Human1Av2 arrays (Agilent Technologies, Palo Alto, CA). 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 was obtained using the Agilent Feature Extraction software (v7.5), using defaults for all parameters.