|
| Status |
Public on Jun 11, 2011 |
| Title |
R1.10 uninfected study2 sample2 |
| Sample type |
RNA |
| |
|
| Source name |
R1.10 hepatoma cells, study 2
|
| Organism |
Homo sapiens |
| Characteristics |
hcv infection susceptibility: very resistant to HCV infection
hcv infected: no
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell cultures were centrifuged at 1000rpm for 5 minutes and the cell pellet was resuspent in 350 microlitre lysis buffer (Qiagen). Each lysate was homogenised with a Qiashedder column (Qiagen) and the RNA was extracted using the RNeasy Mini Kit following the manufactures instructions. On-column DNA digestion was carried out by means of the RNase-Free DNase Set (Qiagen) and the integrity of the RNA was confirmed via Agilent (RIN of 9.7-10).
|
| Label |
biotin
|
| Label protocol |
100ng of RNA from each sample was used to prepare cDNA with the Affymetrix GeneChip 3’ IVT Express Kit and hybridised to Affymetrix U133 Plus 2 microarrays following the manufacturer's instructions
|
| |
|
| Hybridization protocol |
100ng of RNA from each sample was used to prepare cDNA with the Affymetrix GeneChip 3’ IVT Express Kit and hybridised to Affymetrix U133 Plus 2 microarrays following the manufacturer's instructions. The washing and staining procedure was performed in the Affymetrix Fluidics Station 450. The probe array was exposed to 10 washes in 6xSSPE-T at 250C followed by 4 washes in 0.5xSSPE-T at 500C. The biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate, final concentration 2 mg/ml (Molecular Probes, Eugene, OR) in 6xSSPE-T for 30 min at 250C followed by 10 washes in 6xSSPE-T at 250C An antibody amplification step followed using normal goat IgG as blocking reagent, final concentration 0.1 mg/ml (Sigma) and biotinylated anti-streptavidin antibody (goat), final concentration 3 mg/ml (Vector Laboratories). This was followed by a staining step with a streptavidin-phycoerythrin conjugate, final concentration 2 mg/ml (Molecular Probes, Eugene, OR) in 6xSSPE-T for 30 min at 250C and 10 washes in 6xSSPE-T at 250C.
|
| Scan protocol |
The probe arrays were scanned at 560 nm using a confocal laser-scanning microscope (Affymetrix Scanner 3000 7G). CEL files were generated and used for further analysis. All microarray procedures were done at AROS, Denmark.
|
| Data processing |
All analysis of microarray intensity data was carried out using R statistical software including Bioconductor packages. GeneChip probe sets definitions were assigned using Entrez gene version 12.1 of a custom chip description file (CDF) from Psychiatry/MBNI Microarray Lab. This CDF included 17726 probe sets that correspond to an NCBI gene. Initial quality checks of each chip were carried out using Bioconductor core tools and package affyQCReport. Quality checks and visual inspection of array intensities showed that the quality of the array data was good. Robust Multichip Average (RMA) expression values were computed using the Bioconductor affy package. Genes that were called not present on all 39 GeneChip array data sets using Microarray Suite version 5.0 (MAS5) presence calls were removed from the analysis, leaving a total of 13760 genes. Probability, false discovery rate (FDR) and fold-change values for differential expression of genes between cells, using three biological replicates from each, were calculated in a pairwise manner using the limma method in the R limma package. Genes were defined to be significantly differentially expressed if they achieved an FDR of <0.01 and a minimum fold-change of >1.5.
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| |
|
| Submission date |
Jun 10, 2011 |
| Last update date |
Jun 29, 2011 |
| Contact name |
David Robertson |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Manchester
|
| Department |
Faculty of Life Sciences
|
| Street address |
Michael Smith Building
|
| City |
Manchester |
| ZIP/Postal code |
M13 9PT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10526 |
| Series (1) |
| GSE29889 |
Gene expression of a variety of Huh-7 derived hepatoma cells and their susceptibility to infection by JHF1 HCV in the HCV cell culture system. |
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