|
| Status |
Public on Aug 20, 2009 |
| Title |
COH-exposed RGCs4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Purified COH-exposed RGCs
|
| Organism |
Rattus norvegicus |
| Characteristics |
COH-exposed RGCs were isolated according to the two-step immunopanning method reported earlier to yield 95–99% enrichment for RGCs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Purified cells were used for RNA isolation by Stratagene Absolutely RNA Nanoprep kit.
|
| Label |
Cy5
|
| Label protocol |
Target RNA amplification and labeling with Cy5 dye from CyDye Post Labelling Reactive Dye Pack (Amersham, USA) was carried out in two rounds using the Amino Allyl MessageAmp™ aRNA Kit (Ambion, USA) as specified by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
Universal Rat Reference RNA
|
| Organism |
Rattus norvegicus |
| Characteristics |
Comprised as a collection of RNA pooled from fourteen rat cell lines for broad gene coverage, the Universal Rat Reference RNA provides the ability to cross-compare data sets from multiple experiments as a single, common control.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were used for RNA isolation by Stratagene RNA kit.
|
| Label |
Cy3
|
| Label protocol |
Target RNA amplification and labeling with Cy3 dye from CyDye Post Labelling Reactive Dye Pack (Amersham, USA) was carried out in two rounds using the Amino Allyl MessageAmp™ aRNA Kit (Ambion, USA) as specified by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
Once purified and fragmented, the dye labeled aRNA was used for micro-array hybridization. Labeled and amplified RNA from were hybridized to the Oligo microarray (Agilent Technologies) according to the manufacturer’s instructions.
|
| Scan protocol |
The microarrays were scanned at 5 μm resolution using a GenePix 4100A scanner (Axon Instruments at Molecular Devices) and the resulting images were analyzed with the software package GenePix Pro 5.1 (Axon Instruments at Molecular Devices).
|
| Description |
Equivalent amounts of reference and experimental aRNAs were hybridized with the Agilent Rat Oligo Microarrays (Agilent, USA) according to the manufacturer’s instructions.
|
| Data processing |
Data extracted from the images were transferred to the software package Acuity 4.0 (Axon Instruments) for normalization and statistical analysis. Each array was normalized for signal intensities across the whole array and locally, using Lowess normalization. For further analysis genes were selected according to the following quality criteria: (1) at least 90% of the pixels in the spot had intensity higher than background plus two standard deviations; (2), there were less than 2% saturated pixels in the spot; (3) signal to noise ratio (defined as ratio of the background subtracted mean pixel intensity to standard deviation of background) was 3 or above for each channel; (4) the spot diameter was between 25 and 75 μm; (5) the regression coefficient of ratios of pixel intensity was 0.6 or above.
|
| |
|
| Submission date |
Mar 20, 2009 |
| Last update date |
Mar 23, 2009 |
| Contact name |
Dmitry Ivanov |
| E-mail(s) |
divanov@med.miami.edu
|
| Phone |
305-326-6344
|
| Organization name |
University of Miami
|
| Street address |
1638 NW 10th Ave
|
| City |
Miami |
| State/province |
FL |
| ZIP/Postal code |
33136 |
| Country |
USA |
| |
|
| Platform ID |
GPL4135 |
| Series (1) |
| GSE15332 |
Proteomics and transcriptomics analysis of rat retinal ganglion cells exposed to chronic ocular hypertension. |
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