tissue: Grade 3 endometrioid adenocarcinoma - low differentiation genotype: p53 large deletion - null phenotype ip antibody: IgG [Santa Cruz Biotechnology, normal rabbit IgG, catalog # sc-2027]
Treatment protocol
Hec50co cells were maintained in culture without treatment or with 100nM BEZ treatment prior to RIP procedures.
Growth protocol
Hec50co cell lines were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco BRL) at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
Extracted molecule
total RNA
Extraction protocol
The Magna RIP Kit (Millipore, MA, USA) was used to immunoprecipitate MTDH and IgG and isolate immunoprecipitated RNA, and was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Microarray hybridizations were performed at the University of Iowa DNA Facility. Briefly, 25 nanograms total RNA was converted to SPIA amplified cDNA using the WT-Ovation Pico RNA Amplification System, v1 (NuGEN Technologies, San Carlos, CA, Cat. #3300) according to the manufacturer’s recommended protocol. The amplified SPIA cDNA product was purified through a QIAGEN QIAquick PCR Purification column (QIAGEN Cat #28104) according to modifications from NuGEN. Four ug of SPIA amplified DNA were used to generate ST-cDNA using the WT-Ovation Exon Module v1 (NuGEN Technologies, Cat #2000) and again cleaned up with the Qiagen column as above. Five micrograms of this product were fragmented (average fragment size = 85 bases) and biotin labeled using the NuGEN FL-Ovation cDNA Biotin Module, v2 (NuGEN Technologies, Cat. #4200) per the manufacturer’s recommended protocol.
Hybridization protocol
Biotin-labeled cDNA was mixed with Affymetrix eukaryotic hybridization buffer (Affymetrix, Inc., Santa Clara, CA), placed onto Affymetrix Human Exon 1.0 ST array, and incubated at 45º C for 18 h with 60 rpm rotation in an Affymetrix Model 640 Genechip Hybridization Oven. Following hybridization, the arrays were washed, stained with streptavidin-phycoerythrin (Molecular Probes, Inc., Eugene, OR), signal amplified with antistreptavidin antibody (Vector Laboratories, Inc., Burlingame, CA) using the Affymetrix Model 450 Fluidics Station.
Scan protocol
Arrays were scanned with the Affymetrix Model 3000 scanner with 7G upgrade and data were collected using the using the GeneChip operating software (GCOS) v1.4.
Description
RNA-binding protein Immunoprecipitated RNA (RIP) 02_ConigG-2_10-21-10_S2.CEL
Data processing
The Partek Genomics Suite was used to perform microarray data analysis and to generate graphics (Partek GS, St. Louis, MO). Affymetrix array raw fluorescence intensity measures of gene expression were normalized and quantified using robust multi-array analysis. To identify genes differentially expressed between groups, we employed an ANOVA between the groups of interest.
