|
| Status |
Public on Dec 30, 2010 |
| Title |
251209749862 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Universal Human Control RNA
|
| Organism |
Homo sapiens |
| Characteristics |
source: RNA from human cell lines
|
| Treatment protocol |
Samples were peripheral blood drawn from MZ twins and related controls into Qiagen PAXtubes to preserve RNA for later extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the PAXgene RNA Isolation Kit (Qiagen, Inc., Valencia, CA) according to manufacturer's recommendations.
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA was amplified and labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit, according to manufacturer’s protocol.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from whole blood
|
| Organism |
Homo sapiens |
| Characteristics |
sample id: 85-1
disease status: Twin Proband
disease subtype: RA
family: Twin Proband Family 85
|
| Treatment protocol |
Samples were peripheral blood drawn from MZ twins and related controls into Qiagen PAXtubes to preserve RNA for later extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the PAXgene RNA Isolation Kit (Qiagen, Inc., Valencia, CA) according to manufacturer's recommendations.
|
| Label |
Cy5
|
| Label protocol |
500 ng of total RNA was amplified and labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit, according to manufacturer’s protocol.
|
| |
|
| |
| Hybridization protocol |
For each two-color comparison, 750 ng of each Cy3- (universal control) and Cy5-labeled sample cRNA were mixed and fragmented using the Agilent in situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven according to the Agilent 60-mer oligo microarray processing protocol.
|
| Scan protocol |
Slides were scanned using an Agilent Scanner (Agilent Technologies, Wilmington, DE).
|
| Data processing |
Microarray data were obtained using the Agilent Feature Extraction software (v7.5), using defaults for all parameters. The Feature Extraction Software performs error modeling. Raw expression intensities were background subtracted and loess normalized using Agilent Feature Extraction software (v7.5). Normalized log ratios (ratio for each probe was calculated by dividing sample intensity by the universal control intensity followed by log2 transformation) were used for Principal Component Analysis (PCA) to investigate the effect of known sample variables such as subject age, gender, race, disease status, disease activity, medications, and peripheral white blood cell counts and differentials, as well as technical variables such as sample hybridizations performed at different times. We observed no obvious differences across the first three principal components due to age, gender, ethnicity, disease status, or treatment. However, we noticed that samples were segregating in groups represented by their hybridization batch (data not shown). To correct for this difference, we centered the ratios for each probe using the median for their hybridization group. The PCA plots generated using the corrected data showed no obvious groupings; hence we used this data for further analyses. before data are loaded into a database system. Images and GEML files, including error and p-values, were exported from the Agilent Feature Extraction software and deposited into Rosetta Resolver (version 5.0, build 5.0.0.2.48) (Rosetta Biosoftware, Kirkland, WA). The Rosetta Resolver system combines data hybridizations using an error-weighted average that adjusts for additive and multiplicative noise. Resulting log ratios were exported for further analysis.
|
| |
|
| Submission date |
Sep 09, 2010 |
| Last update date |
Dec 30, 2010 |
| Contact name |
Rick David Fannin |
| E-mail(s) |
fannin@niehs.nih.gov
|
| Phone |
919-541-0992
|
| Fax |
919-541-1506
|
| URL |
http://www.niehs.nih.gov/nct/
|
| Organization name |
The National Institute of Environmental Health Sciences
|
| Department |
Office of the Director
|
| Lab |
National Center for Toxicogenomics/Toxicology-Pathology Working
|
| Street address |
111 T.W. Alexander Drive
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL7264 |
| Series (1) |
| GSE24060 |
Peripheral Blood Cells Gene Expression Profiles from Discordant Monozygotic Twins with Systemic Autoimmune disease (SAIDs) |
|
