Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

RNA-binding IMPs promote cell adhesion and invadopodia formation

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To identify factors and pathways regulated by IMP proteins and obtain leads to the mechanism behind the phenotypic changes, we compared the gene expression profiles of IMP siRNA treated cells with mock treated cells. Triplicate gene expression profiles were generated from both the IMP(1,3)A and IMP(1,3)B siRNA sets and were compared to the mock transfected cells. cRNA was hybridized to Affymetrix human U133A arrays.

RNA interference

RNAi knock down of RNA binding IMP proteins in human epithelial (HeLa) cells

To identify factors and pathways regulated by IMP proteins and obtain leads to the mechanism behind the phenotypic changes, we compared the gene expression profiles of IMP siRNA treated cells with mock treated cells. Triplicate gene expression profiles were generated from both the IMP(1,3)A and IMP(1,3)B siRNA sets and were compared to the mock transfected cells. cRNA was hybridized to Affymetrix human U133A arrays.


Affymetrix:Protocol:Hybridization-EukGE-WS2v3

Title: Affymetrix EukGE-WS2v3 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-17351

Six independent experiments with RNAs from mock transfected cells and from cells treated with either IMP(1,3)A and IMP(1,3)B siRNA sets were performed. Three biological replictes were analyzed for each of the three conditions, each replicate being a pool of two mock, IMP(1,3)A or IMP(1,3)B transfected cells, respectively. 

P-MEXP-17353

Human HeLa cells were obtained from the American Type Culture Collection and routinely cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 mg/ml streptomycin. One day before transfection 9000 cells/cm2 were plated and siRNA transfections were performed using Oligofectamine (Invitrogen) and a final concentration of 20 nM siRNA duplex according to the manufacturer's instructions. The media were changed 24 hours after transfection and the cells were examined after another 48 hours. 

P-MEXP-17354

HeLa cells were transfected with siRNAs to knock-down the present IMP proteins. siRNAs were from Qiagen. A BLAST search of the sequences was carried out against GenBank using the NCBI website. Only siRNA duplexes with a similarity region to other human sequences shorter than 15 base pairs were used. The following target sequences were used in this study: IMP1(1), 5'-AAGCTGAATGGCCACCAGTTG-3';  IMP1(2), 5'-AACACCTGACTCCAAAGTTCG-3'; IMP3(1), 5'-AATCGATGTCCACCGTAAAGA-3'; IMP3(2), 5'-AATCCAGAACGCACTATTACA-3'. The IMP siRNAs were used in sets to knock-down both IMP1 and IMP3, and were denoted IMP(1,3)A consisting of the IMP1(1) + IMP3(1)  siRNAs and IMP(1,3)B consisting of the IMP1(2) + IMP3(2) siRNAs. 

P-MEXP-17356

Total RNA was isolated from cells with Trizol reagent (Invitrogen)according to standard protocol.

P-MEXP-17357

. Five micrograms of purified RNA was used to synthesize double-stranded cDNA with the Superscript Choice system (Invitrogen) with an oligo(dT) primer containing a T7 RNA polymerase promoter (GenSet). The cDNA was used as the template for an in vitro transcription reaction to synthesize biotin-labeled antisense cRNA (BioArray high yield RNA transcript labeling kit; Enzo Diagnostics). After fragmentation at 94°C for 35 min in fragmentation buffer (40 mM Tris, 30 mM magnesium acetate, 10 mM potassium acetate), the labeled cRNA was hybridized for 16 h to Affymetrix HG-U133A arrays (Affymetrix Inc.). The arrays were washed and stained with phycoerythrin-streptavidin (SAPE) using the Affymetrix Fluidics Station 400, and the arrays were scanned in the Affymetrix GeneArray 2500 scanner.

P-MEXP-17366

Image scanning was performed using the Affymetrix GeneArray 2500 scanner and the raw values are stored in dat-files. The pixel intensities stored in the raw data dat files were backround corrected and averaged for each probe cell; and the corrected data was stored in cel files using the MicroArray Suite 5 (MAS5) software from Affymetrix. The normalization and expression index summarization and outlier detection were performed with the dChip algorithm, as described (Li and Wong). Briefly, the cel files were imported into the dChip software package and the array files were normalized using the multi array invariant-set normalization method (Li and Wong). All arrays in the experiment were normalized to the “Mock_1a_A” array, which had the median overall intensity of 239. The normalization is based on probes (invariant set) that have similar rank in the baseline and experiment array. Model-based expression values were summarized using the PM-only model implemented in dChip(Li and Wong).

RNAi knock down of RNA binding IMP proteins in human epithelial (HeLa) cells

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