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Sample GSM758821 Query DataSets for GSM758821
Status Public on Jul 13, 2011
Title RIP using MTDH antibody - untreated, biological replicate 1
Sample type RNA
 
Source name Hec50co endometrial cancer cells untreated
Organism Homo sapiens
Characteristics tissue: Grade 3 endometrioid adenocarcinoma - low differentiation
genotype: p53 large deletion - null phenotype
ip antibody: MTDH [Sigma, rabbit, HPA010932]
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)
04_ConMTDH-1_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.
 
Submission date Jul 12, 2011
Last update date Oct 10, 2014
Contact name xiangbing meng
E-mail(s) Xiangbing-meng@uiowa.edu
Organization name University of Iowa
Street address 375 Newton Road
City Iowa City
State/province IA
ZIP/Postal code 52242
Country USA
 
Platform ID GPL5175
Series (1)
GSE30588 Cytoplasmic MTDH associates with mRNAs

Data table header descriptions
ID_REF
VALUE RMA signal intensity

Data table
ID_REF VALUE
3881686 6.97874
2672712 5.99916
2842570 7.44651
3526544 3.31062
2902531 3.74836
2402942 5.12665
3382216 6.18185
3771800 5.96505
2427469 6.11408
2392945 5.43562
2453006 4.23784
3552083 4.70388
3416651 3.91962
2562821 6.38685
3721851 5.41382
3442176 5.56418
2477438 3.10047
2926969 4.6093
3026969 5.42173
3796335 5.44831

Total number of rows: 22011

Table truncated, full table size 341 Kbytes.




Supplementary file Size Download File type/resource
GSM758821.CEL.gz 19.1 Mb (ftp)(http) CEL
Processed data included within Sample table

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