|
| Status |
Public on Feb 14, 2012 |
| Title |
RPEChoroid_34 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Macular RPE-choroid, normal
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: macular RPE-choroid
gender: male
age (years): 63
ocular disease: normal
amd classification: normal
patient identifier: IA073-98
rna integrity number (rin): 5.6
|
| Biomaterial provider |
University of Iowa (G.S.H.)
|
| Growth protocol |
RPE-choroid and retinal samples were isolated from human eye cups obtained from direct sources at the University of Iowa (G.S.H.), the Lion's Eye Bank of Oregon, or direct sources at the University of California, Santa Barbara. The Iowa eyes were selected from a well-characterized repository of 3,903 donors. Medical and ophthalmic histories, a family questionnaire, blood, and sera, were obtained from the majority of donors. All Iowa eyes were read and classified by retinal specialists using gross pathologic features, and when available, the corresponding fundus photographs and angiograms. Iowa eyes were subsequently graded for AMD subtypes using a modified scheme based on the Rotterdam Grading Scale (Normal: no signs of ocular abnormalities; MD1: hard macular drusen only; MD2: soft distinct drusen only; Dry AMD: soft indistinct or reticular drusen only, soft distinct drusen with pigmentary abnormalities, or soft indistinct or reticular drusen with pigmentary abnormalities; CNV: sub-retinal choroidal neovascularization; GA: sharply demarcated area of apparent absence of the RPE (>175 mm) involving central macular region; GA/CNV: geographic atrophy with choroidal neovascularization). Eight mm macular trephine punches and 6 mm temporally adjacent extramacular trephine punches of the RPE-choroid and retina were collected from Iowa eyes within 4 hr postmortem, flash frozen in liquid N2, and stored at -80 oC. The Oregon and Santa Barbara eyes only received a general classification of AMD based on patient histories supported by ophthalmological records. Detailed sample preparation for these eyes is published in Radeke et al. Exp. Eye Res. 85, 366-380 (2007).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified from the RPE-choroid and retina samples obtained from the University of Iowa ocular tissue repository using a Qiagen RNeasy Miniprep kit and on-column DNAse 1 digestion according to the methods of the manufacturer. Total RNA was purified from samples obtained from the Lion's Eye Bank of Oregon and the University of California, Santa Barbara using a Qiagen RNeasy Miniprep kit according to the methods of the manufacturer. Contaminating DNA was digested off-column with DNase I and the DNA-depleted RNA was repurified using the Qiagen RNeasy Miniprep.
|
| Label |
Cy5
|
| Label protocol |
All probe labeling was carried out using the Agilent Low Input Quick Amp Labeling Kit (Part #: 5190-2306, Version 5.7) according to the methods of the manufacturer using 250 ng of total RNA with the exception that spike-in controls were not used.
|
| |
|
| Channel 2 |
| Source name |
Reference RNA
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: RPE-choroid and retina
disease state: normal and AMD
sample type: pool
|
| Growth protocol |
RPE-choroid and retinal samples were isolated from human eye cups obtained from direct sources at the University of Iowa (G.S.H.), the Lion's Eye Bank of Oregon, or direct sources at the University of California, Santa Barbara. The Iowa eyes were selected from a well-characterized repository of 3,903 donors. Medical and ophthalmic histories, a family questionnaire, blood, and sera, were obtained from the majority of donors. All Iowa eyes were read and classified by retinal specialists using gross pathologic features, and when available, the corresponding fundus photographs and angiograms. Iowa eyes were subsequently graded for AMD subtypes using a modified scheme based on the Rotterdam Grading Scale (Normal: no signs of ocular abnormalities; MD1: hard macular drusen only; MD2: soft distinct drusen only; Dry AMD: soft indistinct or reticular drusen only, soft distinct drusen with pigmentary abnormalities, or soft indistinct or reticular drusen with pigmentary abnormalities; CNV: sub-retinal choroidal neovascularization; GA: sharply demarcated area of apparent absence of the RPE (>175 mm) involving central macular region; GA/CNV: geographic atrophy with choroidal neovascularization). Eight mm macular trephine punches and 6 mm temporally adjacent extramacular trephine punches of the RPE-choroid and retina were collected from Iowa eyes within 4 hr postmortem, flash frozen in liquid N2, and stored at -80 oC. The Oregon and Santa Barbara eyes only received a general classification of AMD based on patient histories supported by ophthalmological records. Detailed sample preparation for these eyes is published in Radeke et al. Exp. Eye Res. 85, 366-380 (2007).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified from the RPE-choroid and retina samples obtained from the University of Iowa ocular tissue repository using a Qiagen RNeasy Miniprep kit and on-column DNAse 1 digestion according to the methods of the manufacturer. Total RNA was purified from samples obtained from the Lion's Eye Bank of Oregon and the University of California, Santa Barbara using a Qiagen RNeasy Miniprep kit according to the methods of the manufacturer. Contaminating DNA was digested off-column with DNase I and the DNA-depleted RNA was repurified using the Qiagen RNeasy Miniprep.
|
| Label |
Cy3
|
| Label protocol |
All probe labeling was carried out using the Agilent Low Input Quick Amp Labeling Kit (Part #: 5190-2306, Version 5.7) according to the methods of the manufacturer using 250 ng of total RNA with the exception that spike-in controls were not used.
|
| |
|
| |
| Hybridization protocol |
Probe fragmentation and hybridization was carried out using the Agilent Gene Expression Hybridization Kit (Part #: 5188-5242) according to the methods of the manufacturer (Quick Amp Labeling Protocol, Version 5.7). Arrays were hybridized for 17 hr at 65°C. Arrays were washed for 1 min using Agilent Gene Expression Wash Buffer 1 (Part #: 5188-5325) + 0.005% Triton X-102 at room temperature and for 1 min at 37°C with Agilent Gene Expression Wash Buffer 2 (Part #: 5188-5326) + 0.005% Triton X-102. The washed arrays were then rinsed with acetonitrile for 10 sec and coated with Agilent Stabilization and Drying solution for 30 sec.
|
| Scan protocol |
Scanner: Bio-Rad VersArray™ ChipReader 3mm System. Raw Data Quantification: VersArray™ Analyzer 4.5 [Median raw spot intensity within thresholds (5-65,535), Global Median Background Subtraction (Global background from negative control cells, zero order polynomial approximation), Local Regression Cross-channel Normalization (span = 10%, sub-sampling = 5, single pass)].
|
| Description |
RC_IA073-98 Mac
|
| Data processing |
The data were processed and normalized using a universal reference design. The Lowess normalized raw data were further background corrected by subtraction of the minimum value of the Agilent negative controls (3xSLv1) to obtain the net intensity. Any values less than one standard deviation of the negative control probes were set to a value equal to the standard deviation of the negative controls for that sample. The individual probe net intensity values for both the experimental and reference samples were then divided by the sum of all array probes for that sample and experimental:reference probe ratios were calculated. Each experimental:reference probe ratio was then multiplied by the geometric mean of the corresponding reference probe expressed as percent total of all reference array probes. Values less than the equivalent of the standard deviation of all experimental array negative control spots were set to that equivalent and all values were then multiplied by an arbitrary constant (100,000) so that all expression values were greater than 1. For the two samples with replicates, the average expression values were calculated.
The supplementary files include the raw data (GSMxxx_*_raw.txt) and the data after Lowess normalization and background correction (GSMxxx_*.txt).
|
| |
|
| Submission date |
Jun 07, 2011 |
| Last update date |
Feb 14, 2012 |
| Contact name |
Monte J. Radeke |
| E-mail(s) |
radeke@ucsb.edu
|
| Phone |
805-893-3695
|
| Organization name |
University of California, Santa Barbara
|
| Department |
Neuroscience Research Institute
|
| Lab |
Center for the Study of Macular Degeneration
|
| Street address |
Neuroscience Research Institiute
|
| City |
Santa Barbara |
| State/province |
CA |
| ZIP/Postal code |
93106-5060 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE29801 |
Systems-level analysis of age-related macular degeneration reveals global and subtype-specific functional pathways |
|
