|
| Status |
Public on Sep 27, 2013 |
| Title |
CPE-human-3 |
| Sample type |
RNA |
| |
|
| Source name |
healthy human choroid plexus
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: choroid plexus epithelium
cell sampling: laser dissection microscopy
|
| Treatment protocol |
With laser dissection microscopy, the CPE cells were specifically cut out from cryosections of the choroid plexus (10um) mounted on PEN-membrane slides
|
| 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.
|
| |
|
| Hybridization protocol |
Labeled human CPE RNA and labeled human common reference sample 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 |
reference ch2: tissue: RPE/chodoid, Cy3
CPE replicate 3 of 7 of healthy human choroid plexus
|
| 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 between arrays (method aquantile). In the matrix table, we listed these normalized red channels of each human CPE sample (log2).
|
| |
|
| Submission date |
Aug 19, 2013 |
| Last update date |
Sep 27, 2013 |
| 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 (2) |
| GSE49969 |
Gene expression of the human choroid plexus epithelium (CPE) |
| GSE49974 |
Gene expression of the choroid plexus epithelium (CPE) |
|
