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Sample GSM550700 Query DataSets for GSM550700
Status Public on May 16, 2011
Title medulloblastoma, M2_scrambled, rep3
Sample type RNA
 
Source name medulloblastoma, M2, scrambled
Organism Homo sapiens
Characteristics cell line: DAOY M2.1
cell type: medulloblastoma
genetic modification: siRNA scrambled
Treatment protocol DAOY M2.1 MB cells (70-80% confluent) were transfected using either “SMARTpool siRNA” specific for c-myc or “siCONTROL Non-targeting siRNA Pool” as a control, both purchased from Dharmacon (Thermo Fisher Scientific, Waltham, MA 02454 USA). Each pool of small interfering RNA (siRNA) was used at a final total concentration of 50 nM, in combination with Dharmafect 4 as transfection reagent (Dharmacon), according to the manufacturers’ instructions optimized for adherent cell lines. After 24, 48, and 72 h, cells were harvested for both mRNA and protein extraction to assess gene expression by quantitative real-time PCR (qRT-PCR) and protein content by immunoblotting, respectively.
Growth protocol DAOY cells were obtained as reported elsewhere and maintained in culture as previously described (von Bueren, Shalaby et al. 2007). DAOY cells were grown in GIBCO improved MEM Zinc option 1X, 10% FCS and penicillin-streptomycin. The stable clones DAOY V11 (empty vector-transfected), DAOY M2.1 (c-Myc-overexpressing), and DAOY M14 (c-Myc-overexpressing) (Stearns, Chaudhry et al. 2006) were maintained under selective pressure by supplementing the medium with 500 μg/ml G418.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using the RNeasy columns-Mini Kit (Qiagen, Basel, Switzerland) following the manual’s instructions. After DNA enzymatic digestion (by using RNase-free DNase, Qiagen), 0.5-1 μg of total RNA was used as template for reverse-transcription, triggered by random hexamer primers and performed by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem). Next, qRT-PCR was performed under conditions previously optimized for the ABI7900HT instrument, using Gene Expression Master Mix (Applied Biosystems) and probes/primers specific for c-Myc (Hs00153408_m1), BMP-2 (Hs00154192_m1), BMP-6 (Hs01099594_m1), BMP-7 (Hs00233476_m1) (Applied Biosystems). Normal human cerebellum was used as a reference (Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France).
Label biotin
Label protocol This single-stranded cDNA was fragmented and labeled with biotin using FL-Ovation Biotin Module V2 (NuGEN).
 
Hybridization protocol Biotin-labeled single-stranded cDNA was used for hybridization to GeneChip Human Genome U133 Plus 2.0 arrays for 18h at 45°C. Arrays were then washed using an Affymetrix Fluidics Station 450.
Scan protocol An Affymetrix GeneChip Scanner 3000 was used to measure the fluorescent intensity emitted by the labeled targets. Generated raw data were processed using the Affymetrix AGCC software.
Description Gene expression data.
Data processing Data was preprocessed and analysed with Affymetrix AGCC software. Data were then RMA normalized in R and statistical significance was tested with t-tests (with R).
 
Submission date Jun 04, 2010
Last update date May 16, 2011
Contact name Stefan Zoller
E-mail(s) szoller@env.ethz.ch
Organization name ETH Zurich
Department Genetic Diversity Centre
Street address Universitatstrasse 16
City Zurich
ZIP/Postal code 8092
Country Switzerland
 
Platform ID GPL570
Series (1)
GSE22139 Bone morphogenetic protein-7 is a MYC target with pro-survival functions in childhood medulloblastoma

Data table header descriptions
ID_REF
VALUE Linear RMA-normalized expression signal

Data table
ID_REF VALUE
AFFX-BioB-5_at 338.90
AFFX-BioB-M_at 398.60
AFFX-BioB-3_at 322.70
AFFX-BioC-5_at 760.80
AFFX-BioC-3_at 1013.00
AFFX-BioDn-5_at 1605.00
AFFX-BioDn-3_at 3736.00
AFFX-CreX-5_at 8552.00
AFFX-CreX-3_at 11810.00
AFFX-DapX-5_at 304.60
AFFX-DapX-M_at 642.50
AFFX-DapX-3_at 1213.00
AFFX-LysX-5_at 49.17
AFFX-LysX-M_at 77.99
AFFX-LysX-3_at 170.40
AFFX-PheX-5_at 96.88
AFFX-PheX-M_at 117.10
AFFX-PheX-3_at 163.50
AFFX-ThrX-5_at 102.30
AFFX-ThrX-M_at 163.50

Total number of rows: 54675

Table truncated, full table size 919 Kbytes.




Supplementary file Size Download File type/resource
GSM550700.CEL.gz 4.9 Mb (ftp)(http) CEL
Processed data included within Sample table

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