time (hours): 3 tissue: skin genotype: Wild-type (WT; p53+/+)
Treatment protocol
All mouse treatments have been approved by the institute's Experimental Animal Ethical Committee and carried out in accordance with national legislation. Mice were UVB irradiated at different doses (90, 180, 360, 450, 720 J/m2) in a chamber containing Phillips TL12 lamps. Control mice were mock treated.
Growth protocol
Wild-type (WT; p53+/+) mice were used for UV exposure studies. All mice used were males of 7-10 weeks of age and at least 10 times backcrossed in SKH hairless strain. Mice received normal feed and water ad libitum.
Extracted molecule
total RNA
Extraction protocol
At different time points after UV-B exposure, both treated and untreated mice were anaesthetized by isoflurane. 1.5mm biopsies were sampled from the back. by punching a half moon shape on folded skin. Biopsies were immediately snap frozen in liquid nitrogen and stored at -80°C until further processing. Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA). RNA was assessed for quality with the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
Label
Cy3
Label protocol
Per test sample, 5 µg total RNA combined with 1 µg random octamers (Biolegio) in 4.5 µl was heated to 65°C for 10 min to denature the RNA and was allowed to cool in an ice-waterbath for 10 min. This 4.5 µl was made to 10 µl with a first strand mastermix containing final concentrations of 50 mM Tris-Cl (pH 8.3), 3 mM MgCl2, 75 mM KCl, 200 mM Raffinose (Sigma-Aldrich), 0.015% Triton X-100, 30 ng Actinomycin-D (Sigma-Aldrich), 0.01M DTT, 0.5 mM dGAC, 0.35 mM dUTP, 0.15 mM dUTP-Cy3 (test) or dUTP-Cy5 (common reference) (GE Healthcare) and 200U SuperScript-II (Life Technologies). This mixture was incubated for 2 min at 25°C, 120 min at 42°C and 15 min at 70°C. Finally, 1.5 µl of 2.5M NaOH was added to hydrolyze the remaining RNA by heating for 10 min at 70°C. 8.5 µl 2M MOPS was added for neutralization and the labeled cDNA was purified with the E.Z.N.A. MicroElute RNA Clean-up Kit (Omega Biotek). Dye incorporation and cDNA yield was measured on the NanoDrop ND-1000 (Thermo Scientific) yielding 2-2.5 µg per sample and a FOI > 10 pmol/µg. The common reference was made by an equimolar pool of all test samples (5 µg per sample) and subsequently labeled as the test samples with Cy5 incorporation.
Hybridization protocol
Each hybridization mixture was made up from 750 ng Test (Cy3) and 750 µg Reference (Cy5) sample. Samples were dried and 1.98 µl of water was added. The hybridization cocktail was made according to the manufacturer’s instructions (Nimblegen Arrays User’s Guide – Gene Expression Arrays Version 5.0, Roche Nimblegen). 5.22 µl from this mix was added to each sample. The samples were incubated for 5 min at 65°C and 5 min at 42°C prior to loading. Hybridization samples were loaded onto 12x135k custom designed microarrays against E. coli (OID 38205, Design 120315). Microarrays were hybridized for 18 hours at 42°C with the NimbleGen Hybridization System 4 (Roche Nimblegen).
Scan protocol
the slides were washed according to the Nimblegen Arrays User’s Guide – Gene Expression Arrays Version 5.0 and scanned in an ozone-free room with a Agilent DNA microarray scanner G2565CA (Agilent Technologies). Feature extraction was performed with NimbleScan v2.6 (Roche Nimblegen).
Data processing
The raw data from all arrays was subjected to multiple quality control checks, i.e. visual inspection of the scans, testing against criteria for foreground and background signals, testing for consistent performance of the labeling dyes, checking for spatial effects through pseudo-color plots, and inspection of pre- and post-normalized data with box plots, ratio-intensity (RI) plots and PCA plots. Within array normalization was performed using LOESS. Between array normalization was run on the Cy3-channel by summarizing (Genebank id's) the intensity values of the probes in a probe set using the robust multi-array average (RMA) algorithm.
