|
| Status |
Public on Sep 18, 2012 |
| Title |
PE-5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
healthy human donor eye
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: ciliary body
cell type: PE
cell sampling: laser dissection microscopy
|
| Treatment protocol |
By using laser dissection microscopy, the NPE and PE cells were specifically cut out from the ciliary body cryosection (20um) mounted on PEN-membrane slides
|
| Growth protocol |
n/a
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy Micro Kit (Qiagen Benelux, Venlo, The Netherlands). Subsequently, the mRNA component was amplified with Amino Allyl MessageAmp II aRNA Amplification kit (#1753, Ambion Applied Biosystems Europe B.V.).
|
| Label |
Cy5
|
| Label protocol |
aRNA samples were labeled with a Cy5 fluorescent probe (Cy5 mono-reactive dye pack, GE Healthcare UK, Little Chalfont, Buckinghamshire, UK). In short, aRNA was coupled to Cy5 (dissolved in DMSO) in 0.5 M NaHCO3 (pH 9.0) for one hour. Free dyes were quenched by the addition of 4 M hydroxylamine and removed by filtration over a Chroma Spin-30 column (DEPC-H20 Columns, Clontech Laboratories) with glycogen as a carrier. Cy3-labeled common reference sample.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from pooled healty human RPE/choroid cells labeled with Cy3
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: RPE/chodoid [common reference]
|
| Treatment protocol |
By using laser dissection microscopy, the NPE and PE cells were specifically cut out from the ciliary body cryosection (20um) mounted on PEN-membrane slides
|
| Growth protocol |
n/a
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy Micro Kit (Qiagen Benelux, Venlo, The Netherlands). Subsequently, the mRNA component was amplified with Amino Allyl MessageAmp II aRNA Amplification kit (#1753, Ambion Applied Biosystems Europe B.V.).
|
| Label |
Cy3
|
| Label protocol |
aRNA samples were labeled with a Cy5 fluorescent probe (Cy5 mono-reactive dye pack, GE Healthcare UK, Little Chalfont, Buckinghamshire, UK). In short, aRNA was coupled to Cy5 (dissolved in DMSO) in 0.5 M NaHCO3 (pH 9.0) for one hour. Free dyes were quenched by the addition of 4 M hydroxylamine and removed by filtration over a Chroma Spin-30 column (DEPC-H20 Columns, Clontech Laboratories) with glycogen as a carrier. Cy3-labeled common reference sample.
|
| |
|
| |
| Hybridization protocol |
Labeled RNA of interest (NPE of PE) and labeled common reference sample togheter with hybridization buffer were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential
|
| Scan protocol |
Scanned on an Agilent scanner, protocol GE2-v5_95_Fec07
|
| Description |
Biological replicate 5 of 7 of PE cells of healthy human ciliary body tissue, same donor as for NPE-5
|
| Data processing |
Agilent Feature Extraction Software (v 9.5.3.1) was used for background subtraction and linear loweless dye normalization. This resulted amongst others in normalized log ratios (test/reference) data that can be find in the Agilent Feature Extraction files.
Further data processing was done in R (version 2.14.0 for Windows, R Development Core Team, 2009), with package Limma. We read in the raw data (RG <-read.maimage). Next we normalized within arrays (method 'loess', resulating in MA) and than we normalized the red signals between arrays to make it possible to compare the red channels (method "q", resulting in MA.s). In the matrix table, we listed these normilized red channels of each NPE and PE sample (log2). These data were used to identify the highly expressed genes of the NPE and PE (based in 90th percentile).
|
| |
|
| Submission date |
Jul 06, 2012 |
| Last update date |
Sep 18, 2012 |
| Contact name |
Sarah Janssen |
| E-mail(s) |
s.janssen@nin.knaw.nl
|
| Organization name |
Netherlands Institute for Neuroscience
|
| Department |
Ophthalmogenetics
|
| Street address |
Meibergdreef 47
|
| City |
Amsterdam |
| ZIP/Postal code |
1105 BA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE37957 |
Gene expression of the pigmented and non-pigmented epithelia of the ciliary body of human healthy eyes |
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