|
| Status |
Public on Dec 31, 2012 |
| Title |
ME-15_pcDNA3.1-SOCS3_IFNa_1000Uml_24h_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
cell_line_ME15-SOCS3-cloneB
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: ME15-SOCS3-cloneB
incubation_time: 24
treatment: Interferon-alpha-2a
treatment_dose_u/ml: 1000
plasmid: pCDNA3.1+NEO SOCS3 B
cell_line_clone: ME3-B
sentrix barcode: 4235991022
sample section: B
|
| Treatment protocol |
Roferon (Interferon alpha2a, ROCHE) was diluted in fresh culture medium to a final concentration of 1,000 U/ml and control cultures were grown without cytokine. Cell cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 4 or 24 hours.
|
| Growth protocol |
ME-15 and descendent cell lines were cultured at 37°C in a 5% CO2 atmosphere in RPMI 1640 medium (GIBCO Life Sciences, Paisley, U.K.) supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential amino acids, antibiotics, and 10 mM HEPES buffer. HuH-7 and descendent cell lines were cultured in D-MEM + GlutaMAX™-I containing 10% FBS (D-MEM culture medium). All cell culture reagents were purchased from Invitrogen (GIBCO®).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from independent triplicates using the Trizol reagent (Sigma) as described previously (Siegrist F et al. 2009 Biol Proced Online 11(1):113-129). RNA was quantified using the Quant-iT™ RiboGreen® RNA Assay (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
500 ng total RNA subjected to cDNA synthesis and subsequent in vitro transcribed to biotin labeled cRNA using the Illumina® TotalPrep RNA Amplification Kit (Ambion). cRNA was quantified with QuantIT against ribosomal RNA standard (Invitrogen).
|
| |
|
| Hybridization protocol |
750 ng of biotin labeled, intact cRNA was hybridized to Sentrix HumanRef-8 v3 Expression BeadChips (Illumina) according to the manufacturer’s protocol. Replicates were randomly distributed among slides and positions. Bead arrays were washed and stained using FluoroLink Cy3 Streptavidin (GE Healthcare Life Sciences).
|
| Scan protocol |
Fluorescent signal data was collected using a high-resolution scanner system (Illumina iScan) using factory settings for whole genome expression arrays.
|
| Data processing |
Scanner images files were processed by software supplied from the manufacturer to probe intensity files and further processed with the GenomeStudio software, gene expression module 1.0.6 (Illumina, Inc.) without normalization and background correction. VST (variance stabilization) transformation 'lumiT()', quantile normalization 'lumiN(, method = quantile)' and statistical analysis were performed with R/Bioconductor software (2.11.1) using the package lumi (1.14.0).
|
| |
|
| Submission date |
Jul 07, 2010 |
| Last update date |
Dec 31, 2012 |
| Contact name |
Fredy Siegrist |
| E-mail(s) |
fredy.siegrist@dkf.unibe.ch
|
| Organization name |
University Hospital Bern
|
| Department |
Department of Nephrology
|
| Lab |
KKL D830
|
| Street address |
Freiburgstrasse 15
|
| City |
Bern |
| ZIP/Postal code |
3010 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL6883 |
| Series (1) |
| GSE22801 |
Interferon alpha induced gene expression in SOCS1 and SOCS3 overexpressing melanoma and hepatoma cell lines. |
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