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

Transforming activity of MECT1-MAML2 fusion oncoprotein is mediated by constitutive CREB activation

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Identification of down-down target genes differentially regulated by the MECT1-MAML2 oncoprotein, as compared to two translocation gene partners, MAML2 and MECT1.

transfection

Transcription profiling of HeLa cells transfected with MECT1-MAML2_Targets

Identification of down-down target genes differentially regulated by the MECT1-MAML2 oncoprotein, as compared to two translocation gene partners, MAML2 and MECT1.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4[]

Title: Affymetrix EukGE-WS2v4 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-8439

HeLa Tet-On (Clontech) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% inactivated fetal calf serum and penicillin/streptomycin. Cells were grown at 37°C under 5% CO2.

P-MEXP-8440

HeLa Tet-On cells were transfected with the pRevTRE vectors expressing bicistronic message RNAs for both HA tagged MAML2 (or MECT1, or MECT1-MAML2) and GFP, and induced for expression in the presence of 200 ng/ml Doxycyclin for 24 h.

P-MEXP-8442

GFP positive cells were sorted, and the total RNA extracted using Trizol reagents (Life Technologies) and purified via the columns from SV 96 Total RNA Isolation kit (Promega).

P-MEXP-8443

RNA is converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis is then carried out. The double-stranded cDNA is used as the template in an in vitro transcription (IVT) reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphates. The biotinylated complementary RNA (cRNA) is purified from the IVT reaction mixture using the RNeasy system (Qiagen). The cRNA is quantified spectrophotometrically and purity of the cRNA is also assessed by spectrophometric measurements. Those cRNAs that fall outside of an acceptable range will not be carried forward in the analysis. The purified cRNA is fragmented in order to facilitate the subsequent hybridization step.

P-MEXP-8444

The cRNA is purified from the fragmentation reaction using phenol/chloroform extraction and ethanol precipitation. The fragmented cRNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C. The chips are then transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.

Hela_MM2_scan

Each cRNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis.

P-MEXP-8571

The global scaling (all probe sets) method recommended by Affymetrix was used. For a detailed description, please see the Affymetrix website.

Transcription profiling of HeLa cells transfected with MECT1-MAML2_Targets

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