|
| Status |
Public on Jun 17, 2011 |
| Title |
Normal5_young_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
healthy fibroblast
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: fibroblast
disease state: healthy
cell culture passage: early
treated with tert: yes
individual: normal 5
|
| Growth protocol |
Cells were grown under 5% CO2 in MEM supplied by 15 % FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from 1 million cells with the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. Total RNAs were prepared according to Affymetrix protocols (Affymetrix, Inc). RNA quality and quantity was ensured using the Bioanalyzer (Agilent, Inc) and NanoDrop (Thermo Scientific, Inc), respectively.
|
| Label |
biotin
|
| Label protocol |
For RNA labeling, 1 microgram of total RNA was used in conjunction with the Affymetrix recommended protocol for the GeneChip® WT Sense Target Labeling kit. The total RNA was first subjected to a ribosomal RNA reduction using the Invitrogen RiboMinus Transcriptome Isolation Kit. The remaining RNA was then reverse transcribed in the presence of dT7N6 primers to generate cDNA. An in vitro transcription was then performed and the resulting cRNA was purified. A first strand cDNA was generated form the cRNA in the presence of dUTPs. After RNA hydrolysis, the single stranded cDNA was fragmented and end labeled with Biotin.
|
| |
|
| Hybridization protocol |
The hybridization cocktail containing the fragmented and Biotin labeled cDNAs were hybridized to The Affymetrix GeneChip® Human Exon 1.0 ST Array. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols as described by Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Molecular Probes, Carlsbad, CA) and enhanced by using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA). Each sample was processed in technical duplicates or triplicates.
|
| Scan protocol |
An Affymetrix Gene Chip Scanner 3000 was used to scan the probe arrays. Gene expression intensities were calculated using the Affymetrix Expression Console software and the CEL files were generated.
|
| Description |
KC_04-8398T-P8
|
| Data processing |
RMA expression values were derived from Expression Console software, and all downstream exon array analyses were performed using XRAY microarray analysis software, Version 3.9. Full quantile normalization was performed to minimize technical variance within the data. Only ‘Core’ probe-sets (probe-sets corresponding to high quality genomic features like RefSeq or Ensembl transcripts) were considered for analysis. Probes with GC count less than 6 and greater than 17 were filtered from the analysis and the probe scores were transformed by taking natural logarithm of 0 plus the probe score. Background correction for each probe score was carried out by subtracting the median expression score of background probes with similar GC content. The expression score for a probe-set was calculated as the median of all probes within the set, and probe-sets with fewer than three probes were filtered from further analysis. Mixed Model, nested ANOVA, was used to identify genes with statistically significant group specific gene expression or alternative splicing. The FDR method was used to correct for multiple testing and FDR of 1.00E+00 or less was considered significant both for differential alternative splicing and gene expression tests. A p-value threshold of 0.001 was used to reject the null hypothesis that the average of group expression is not above background. P-value filters were also applied to group expression levels for each gene to reduce false-positives for alternative splicing. Quality Control diagnostics produced for each array before and after normalization proved the good quality of arrays. GO enrichment analysis for the gene lists was carried out using MetaCore (MetaCoreTM, GeneGo, St. Joseph, MI). Categories with an FDR of 0.05 or below were considered statistically significant.
Core exon analysis.
|
| |
|
| Submission date |
Apr 28, 2011 |
| Last update date |
Jun 17, 2011 |
| Contact name |
Kan Cao |
| E-mail(s) |
kcao@umd.edu
|
| Organization name |
University of Maryland
|
| Department |
CBMG
|
| Street address |
2219 Bioscience Research building
|
| City |
College Park |
| ZIP/Postal code |
20742 |
| Country |
USA |
| |
|
| Platform ID |
GPL5175 |
| Series (1) |
| GSE28863 |
Exon array analysis in primary human fibroblasts |
|
