|
| Status |
Public on May 12, 2009 |
| Title |
PBLs_pair 220263_CFS |
| Sample type |
RNA |
| |
|
| Source name |
PBLs, pair 220263, CFS
|
| Organism |
Homo sapiens |
| Characteristics |
twin pair: 220263
sex: female
diagnonsis: CFS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Peripheral venous blood was drawn using sterile technique into PAXgene tubes manufactured in the same batch (Qiagen, to protect RNA from degradation and to minimize ex vivo gene expression). Total RNA was purified using the PAXgene blood RNA kit following the manufacturer’s instructions (Qiagen). RNA quality was determined using the Agilent 2100 Bioanalyzer.
|
| Label |
biotin
|
| Label protocol |
Total RNA (5.0 μg) was labeled with the one-cycle cDNA synthesis kit (Invitrogen) and spiked with eukaryotic Poly-A RNA controls to check the target labeling process (Affymetrix). Synthesized cDNA was transcribed in vitro using the GeneChip IVT labeling kit (Affymetrix). The biotin labeled cRNA product (20 μg) was purified with a sample cleanup module (Qiagen) and samples were fragmented with the fragmentation buffer from Affymetrix at 94°C for 35 minutes.
|
| |
|
| Hybridization protocol |
Fragmented and labeled targets (together with hybridization and oligo B2 controls) were hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays at 45°C for 16 hours. Washing and staining of the arrays were performed on the Affymetrix fluidics station using the EukGE-WS2v5_450 protocol.
|
| Scan protocol |
Imaging of the arrays and signal quantification were performed with the Affymetrix GeneChip Scanner 3000 and GeneChip Operating Software. For verification, we used qRT-PCR. RNA was converted to cDNA with Superscript III (Invitrogen) and qRT-PCR was run with ABI's Taqman gene expression assays (with 18S rRNA as control). The ΔΔCt method was used for the calculations.
|
| Description |
gene expression data from PBLs, pair 220263, CFS, female
|
| Data processing |
Array images were manually checked for defects using DChip and then normalized using the RMA algorithm in Affymetrix Expression Console (v1.0). After normalization, the Bioconductor Significance Analysis of Microarrays package was used to compute modified paired t-tests that contrasted an affected twin with the unaffected co-twin for each transcript using R. To adjust for multiple comparisons, the nominal permutation-based p-values from SAM were used to compute false discovery rate q-values.
|
| |
|
| Submission date |
May 12, 2009 |
| Last update date |
May 12, 2009 |
| Contact name |
Patrick F Sullivan |
| E-mail(s) |
pfsulliv@med.unc.edu
|
| Organization name |
UNC Chapel Hill
|
| Department |
Genetics
|
| Street address |
4109D Neuroscience Research Building, 103 Mason Farm Road
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599-7264 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE16059 |
Gene Expression in Peripheral Blood Leucocytes in Monozygotic Twins Discordant for Chronic Fatigue |
|
