|
| Status |
Public on May 01, 2010 |
| Title |
Huh7 cells_Mock Infected 12 hours_ rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Huh7 cells, Mock Infected, 12 hours.
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: hepatoma cells
cell line: Huh7
|
| Treatment protocol |
5x105 Huh7 cells were seeded in 25cm2 culture flasks and infected in triplicate either with the genotype 2a HCV clone, JFH-1 at a multiplicity of infection (MOI) of 3 or mock infected with an equal volume of concentrated conditioned growth medium
|
| Growth protocol |
Huh7 cells: Huh7 cells were grown in Dulbecco’s modified Eagles Medium (DMEM) supplemented with 10% foetal calf serum, 100U/ml penicillin and 100µg/ml streptomycin (Invitrogen) at 37°C, 5% CO2. HCV JFH-1 production: Full-length JFH-1 virus was produced using Huh7.5 cells transfected with RNA transcribed from the pJFH-1 plasmid. Transfected cells were maintained for 21 days and growth medium containing infectious JFH-1 was serially passaged onto naive Huh7.5 cells every four days. Culture medium containing virus was harvested at each passage, clarified by centrifugation and 0.45µm filtration, and concentrated using Amicon Ultra-15 centrifugal filter units (Millipore).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
3ml of TRIzol reagent (Invitrogen) was added to the cells in each T25 flask. RNA was extracted according to the manufacturer’s protocol.
|
| Label |
Biotin
|
| Label protocol |
Total RNA was first reverse transcribed using a T7-Oligo(dT) Promoter Primer in the first-strand cDNA synthesis reaction. Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA was purified and served as a template in the subsequent in vitro transcription (IVT) reaction. The IVT reaction was carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling.The biotinylated cRNA targets were then cleaned up, fragmented, and hybridized to GeneChip expression arrays. Quality of labeled RNA was assessed using the Agilent Bioanalyser.
|
| |
|
| Hybridization protocol |
Hybridisation of cRNA was performed using a hybridisation cocktail of 15ug cRNA, 50pM control oligonucleotide B2, Eukaryotic hybridisation controls (1.5pM bioB, 5pM bioC, 25pM bioD and 100pM cre), 0.1mg/ml Herring sperm DNA, 0.5mg/ml BSA, 1x hybridisation buffer and 10% DMSO. The hybridisation cocktail was heated to 99°C for 5 minutes followed by 45°C for 5 minutes. The array was filled with 1x hybridisation buffer and heated to 45°C for 10 minutes. Hybridisation buffer was removed from the array and the hybridisation cocktail was added and incubated for 16 hours at 45°C. Following hybridisation, arrays were washed using both non-stringent and stringent wash buffers. The array was then stained using 10ug/ml streptavidin phycoerythrin diluted in stain buffer with 2mg/ml BSA for 10 minutes, followed by incubation with goat anti-streptavidin biotinylated antibody diluted in stain buffer containing 2mg/ml BSA and 0.1mg/ml goat IgG stock for 10 minutes and a second round of staining with 10ug/ml streptavidin phycoerythrin diluted in stain buffer with 2mg/ml BSA for 10 minutes.The array was washed again with non-stringent wash buffer and was placed onto the scanner.
|
| Scan protocol |
The stained array chips were scanned using the Affymetrix genechip scanner 3000 which was operated using the Affymetrix GeneChip Operating System (GCOS).
|
| Description |
Gene expression data from Huh7 cells treated with mock media for 12 hours
|
| Data processing |
Raw microarray expression data was analyzed with Array Studio software (Omicsoft, USA) using MicroArray data default settings and RMA signal extraction method. Two samples (Mock_6hrs_1 and JFH-1_6hrs_1) did not pass our data quality control measures and were excluded from the analysis. Data was analyzed on the log2 scale, using analysis of variance (ANOVA), followed by pre-specified contrast statements. Fold changes were obtained from the estimated least squares mean differences. Host genes demonstrating at least a 2 fold change in expression, a significant differential expression at a raw p-value level of 0.05, and an intensity level above 32 (5 on the log2 scale) for at least one of the treatment means were selected for further investigation.
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| |
|
| Submission date |
Mar 18, 2010 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Michael J McGarvey |
| E-mail(s) |
m.mcgarvey@imperial.ac.uk
|
| Phone |
+44 (0)20 594 9035
|
| Organization name |
Imperial College London
|
| Department |
Medicine
|
| Street address |
Norfolk Place
|
| City |
London |
| ZIP/Postal code |
W2 1PG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE20948 |
The Effect of Hepatitis C Virus Infection on Host Gene Expression |
|
| Relations |
| Reanalyzed by |
GSE119087 |
