cell line: Hec50co endometrial cancer cell line cell line traits: Loss of p53, loss of hormone receptors ER and PR disease state: Grade 3 endometrioid adenocarcinoma genotype/variation: control
Treatment protocol
We transfected cells with MTDH short hairpin RNA interference constructs provided from ORIGENE (Rockville, MD). Individual cell clones resistant to puromycin were expanded and subjected to screening of the expression of the targeted protein by Western blotting.
Growth protocol
These 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
mirVana miRNA Isolation Kit (Ambion, Austin, TX) RNA extraction of total RNA 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 Gene 1.0 ST array (Part No. 901085), 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
Gene expression data resulting from the knockdown of MTDH in Hec50co endometrial cancer cells. 5_MTDHplus2_4-13-10_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.
