After hypoxic preconditioning (6% O2 for 6 hours) reoxygenation was allowed in darkness for 0 h, 2 h, 4 h, 12 h and 16 h in normal room air in darkness. After reoxygenation BALB/c mice were exposed to 5'000 lux of white fluorescent light for 1 h and analyzed at the time points as indicated (Thiersch et al, BMC Genomics. 2008, 9:73).
Growth protocol
BALB/c mice were grown under standard conditons (ARVO).
Extracted molecule
total RNA
Extraction protocol
Retinas were isolated immediately, 0 h, 2 h, 4 h and 16 h after hypoxic preconditioning, frozen in liquid nitrogen and stored at -70°C, 3 retinas of 3 different mice were pooled and the procedure repeated 3 times to generate independent biological triplicates. RNA was extracted using the RNeasy isolation kit (Qiagen, Hilden, Germany), including a DNase treatment to digest residual genomic DNA.
Label
biotin
Label protocol
Biotinylated cRNA was prepared from the total RNA according to the Affymetrix protocol (Expression Analysis Technical Manual Rev. 2, 2005-2007, Affymetrix)
Hybridization protocol
Following fragmentation, cRNA was hybridized on the GeneChip Array and the GeneChips were washed and stained in the Affymetrix Fluidics Station 450 according to the standard Affymetrix protocol (Expression Analysis Technical Manual Rev. 2, 2005-2007, Affymetrix).
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip 3000 7G scanner.
