|
| Status |
Public on Jan 01, 2014 |
| Title |
PE_Fgf2_t120h_tech-rep2_Cy3 |
| Sample type |
RNA |
| |
|
| Source name |
PE, Fgf2, t120h
|
| Organism |
Gallus gallus |
| Characteristics |
CSK3_110_PE_Fgf2_t120h_tech-rep2_Cy3
|
| Treatment protocol |
PE explant cultures were treated with either U0126+Bmp2, Fgf2 or Fgf2+Vegf as described by Boudewijn P T et al ( Dev Biol. 2006 Jul 15;295(2):507-22 )
|
| Growth protocol |
Embryonic Proepicardia and Epicardium were isolated and grown as described by van den Hoff MJ et al ( Dev Biol. 2001 Dec 1;240(1):61-76 ) and Boudewijn P T et al ( Dev Biol. 2006 Jul 15;295(2):507-22 )
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Macherey-Nagel nucleospin columns and DnaseI treated. Quantified with the Nanodrop ND1000 and QC with an Agilent nanoChip.
|
| Label |
Cy3
|
| Label protocol |
1500 ng amplified RNA ( Ambion MessageAmp-II ) wasd labelled with either Cy3 or Cy5 as decribed by 't Hoen PA ( Nucleic Acids Res. 2003 Mar 1;31(5):e20 )
|
| |
|
| Hybridization protocol |
Slides were pre-hybridized (5x SSC, 25% (v/v) formamide, 0.1% (w/v) SDS, 1% (w/v) bovine serum albumin, fraction V (Sigma)) for 45 minutes at 42°C to block the remaining reactive epoxide groups and reduce background signal. Cy3 and Cy5 labelled RNA samples were combined as described in the experimental design and mixed with 2x hybridization mixture to a final concentration of 5x SSC, 25% (v/v) formamide, 0.1% (w/v) SDS, 0.2 \mug/\muL herring sperm DNA (Invitrogen). Target-RNA mix was denatured for 5 minutes at 70°C and incubated at 42°C for 30 minutes. Hybridization was done in a GeneTAC hybridization station (Genomic Solutions) for 16 hours at 42°C with agitation. The arrays were washed at room temperature in 5 successive buffers, i.e., 1x SSC + 0.2% SDS, 0.1x SSC + 0.2% SDS, 1x SSC, 0.1x SSC and 0.001x SSC, each step lasting 4 minutes. The slides were finally pressure-air dried and stored in the dark.
|
| Scan protocol |
Slides were scanned using an Agilent G2565BA microarray scanner on a 10 micro m resolution. The resulting images were split into the red and green channels prior to feature extraction with the GenePix Pro 6.1.0.2 (Axon Instruments Inc.) software.
|
| Description |
F2-series_Array-CSK3_110:Cy3-PEt120h|Cy5-PEt72h
|
| Data processing |
Median spot intensities were imported from the genepix results files using the Limma package. Flagged spots were assigned a weight of 0.1, control spots were removed and the remaining data were normalized with lowess (parameters: span = 0.25, iterations = 2) and aquantile transformations using the R/Limma package in Bioconductor
|
| |
|
| Submission date |
Dec 12, 2008 |
| Last update date |
Jan 01, 2014 |
| Contact name |
Henk Buermans |
| E-mail(s) |
h.buermans@lumc.nl
|
| Phone |
+31715268558
|
| Organization name |
Leiden University Medical Center
|
| Department |
Human Genetics
|
| Lab |
Leiden Genome Technology Center
|
| Street address |
Albinusdreef 2
|
| City |
Leiden |
| State/province |
ZH |
| ZIP/Postal code |
2300 RC |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL5480 |
| Series (1) |
| GSE32508 |
Specific Lineage differentiation of proepicardial explant cultures |
|
