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Novel N-terminal Glucocorticoid Receptor Isoforms with Unique Transcriptional Target Genes

ABSTRACT: Gene expression analysis was conducted using Agilent Human1Av2 arrays (Agilent Technologies, Palo Alto, CA). Two separate biological replicates of cytoplasmic RNA samples were harvested and purified from each of the U-2 OS cell lines stably expressing hGRalpha, -A, -B, -C3, or D3 treated with 100 nM DEX or vehicle for 6 hours using RNeasy Midi kits (Invitrogen). These RNA samples were amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 0.5 ng of total RNA, two cRNAs, one labeled with Cy3 and one labeled with Cy5 were produced for each sample according to the manufacturer’s protocol. Consequently, four separate chips were used for each of the isoforms at each treatment condition, yielding a total of 40 chips: 5 isoforms X 2 treatments (vehicle vs. hormone) X 2 biological replicates X 2 labeling dyes. For each two-color 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 16 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 obtained using the Agilent Feature Extraction software (v7.1), using defaults for all parameters. Keywords: other

ORGANISM(S): Homo sapiens

PROVIDER: GSE2428 | GEO | 2005/06/20

SECONDARY ACCESSION(S): PRJNA91587

REPOSITORIES: GEO

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Project description:Surgically resected gastric adenocarcinoma samples and their paired disease-free (R0) marginal nonmalignant tissue were obtained from 14 patients with gastric adenocarcinoma. RNA was isolated using the RNAeasy Kit (Qiagen Valencia, CA) from 15 mg of tissue. The quality of total RNA and its integrity was assessed using the Bioanalyzer 2100 (Agilent, Palo Alto, CA) and RIN value (RNA Integrity Number) was recorded for all the samples for intact 18S and 28S rRNA. Total RNA (800 ng) from each sample was reverse transcribed and linear amplified using the low RNA input linear amplification kit (Agilent Technologies). After synthesis of the first and second strand of cDNA, the product was used in an in vitro transcription reaction to generate cRNAs in the presence of cyanine 3-labeled UTP (Cy3) for normal and cyanine 5-labeled UTP (Cy5) for tumor. Fragmented Cy3-labeled cRNA of the control sample were mixed with equal amounts of Cy5-labeled cRNA from the gastric tumor sample, and the mixtures were hybridized onto 44K whole human genome DNA microarrays (Agilent Technologies) for 17 hrs at 65°C with constant rotation (10 rpm). Subsequently, the arrays were washed according to manufacturer’s instructions. The slides were scanned using an Agilent microarray scanner (G2565AA), and the images were processed using the Agilent feature extraction software AFE 9.5. GeneSpring GX v10.1 (Agilent Technologies) was used to analyze the expression profiles obtained after microarray hybridization. The feature extraction files from all the samples were uploaded to GeneSpring. Following intensity-dependent normalization (Lowess), the data was subjected to statistical analysis. A two tailed student’s t-test was used to determine significance of the differences observed between the normal and tumor samples. Benjamini Hochberg algorithm was used to compute false discovery rates. Genes were filtered based on P value threshold of 0.001 and false discovery rate of less than 1%.

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Novel N-terminal Glucocorticoid Receptor Isoforms with Unique Transcriptional Target Genes

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