|
| Status |
Public on Nov 01, 2007 |
| Title |
HCV infected liver _Fibrosis Stage 0_pool1 |
| Sample type |
RNA |
| |
|
| Source name |
Pooled early fibrosis Stage 0 of chronic HCV infected liver
|
| Organism |
Homo sapiens |
| Characteristics |
Tissue: Liver biopsy tissue
Disease stage: A pooled RNA sample from 4 liver tissues with chronic hepatitis C, fibrosis stage 0, grade of inflammation 0-1.
|
| Biomaterial provider |
All liver biopsy specimens were collected at Aga Khan University & Hospital following institutional ethics committee guidelines, from 3-10-2005 to 10-9-2005
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from frozen liver tissues using TRIzol reagent (Invitrogen, MD) and purified using affinity resin column (Rneasy Qiagen, Chatsworth, CA). Equal amounts of total RNA from 4 individuals with fibrosis stage 0 were pooled. The quality of RNA was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Biotin
|
| Label protocol |
50ng of the isolated total RNA was converted to cDNA and amplified according to the manufacturer’s instructions for the Affymetrix GeneChipTM System (Santa Clara, CA) at the Microarray Facilities, Mayo Foundation General Clinic Research (Rochester, MN). Double stranded cDNA was then purified by phase lock gel (Eppendorf, Westbury, NY) with phenol/chloroform extraction. Purified cDNA was used as a template for an in vitro transcription reaction for the synthesis of biotinylated cRNA using RNA transcript labeling reagent (affymetrix, Inc., Santa Clara, CA).
|
| |
|
| Hybridization protocol |
The labeled cRNAs were then fragmented and hybridized onto the Affymetrix GeneChipTM arrays (Affymetrix HG U133 plus 2.0), according to manufacturer’s instructions. Briefly, appropriate amounts of fragmented cRNA and control oligonucleotide B2 were added to the hybridization buffer along with control cRNA (BioB, BioC, BioD), herring sperm DNA, and bovine serum albumin (BSA). The hybridization mixture was heated at 99o C for 5 min followed by incubation at 45o C for 5 min before injecting the sample into the GeneChipTM. Then the hybridization was done at 45oC for 16 h with mixing on a rotisserie at 60 rpm. After hybridization, the solution was removed and arrays were washed and stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR).
The quality of the fragmented biotin-labeled cRNA in each experiment was evaluated by both gel electrophoresis and hybridization (fraction of the sample) onto test-3 microarray as a measure of quality control before hybridizing onto the Affymetrix Gene Expression Arrays.
|
| Scan protocol |
Probe arrays were scanned in the Affymetrix GeneChipTM system confocal scanner by using Affymetrix GeneChipTM Operating System v1.3 software. Chips were scaled to a value of 500 to allow array-to-array comparisons.
|
| Description |
Experiment ID: Rag208 HG U133Plus 2.0
|
| Data processing |
The CEL files generated by Gene Chip Operating Software (GCOS) were uploaded to Genespring GX software. Gene expression measures were computed using the Robust Multi-chip average method (RMA) that includes background correction, probe-level quantile normalization and calculation of expression measures.
|
| |
|
| Submission date |
Apr 14, 2007 |
| Last update date |
Dec 18, 2019 |
| Contact name |
Saira Khalid |
| E-mail(s) |
[email protected]
|
| Organization name |
King Saud University
|
| Street address |
Amr Ibn Alaas street
|
| City |
Riyadh |
| ZIP/Postal code |
35006 |
| Country |
Saudi Arabia |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE7741 |
Expression data of HCV-associated advance disease state |
|
| Relations |
| Reanalyzed by |
GSE64985 |
| Reanalyzed by |
GSE119087 |
