HaCaT keratinocytes were seeded at 10 000 cells /cm2 and cultured as monolayers in DMEM supplemented with 10% fetal calf serum at 37°C and 5% CO2. At day 7, the culture was confluent, and it was further kept in culture for 3 days. In these conditions, cell density-arrested keratinocytes undergo the differentiation program found in the suprabasal layers of the epidermis (spinous and granular layers). For the microarray studies, 300 cm2 flasks were irradiated at day 3 and 10 with a 60cobalt source at a dose rate of 0,4 Gy/min. A radiation dose of 2 Gy was used. Cells were incubated again after exposure, and cells were harvested at 3 hours after irradiation. Reference cells followed exactly the same treatment but were not irradiated. Total RNA was isolated using the Tripur reagent (Roche Molecular Biochemicals, Meylan, France) following the manufacturer’s instruction and dissolved in RNAse-free water. The quality of RNA was controlled by using an Agilent 2100 Bioanalyser (Agilent technologies, Palo Alto, CA, USA) and concentration was measured by absorbance at 260 nm. Labelling of total RNA was performed by incorporation of aminoallyl-modified dUTP (Sigma, St Quentin-Fallavier, France), followed by a subsequent incorporation of monofunctionnal Cy3 or Cy5-N-succinimide esters (Amersham Biosciences, Orsay, France). For each labelling experiment, 20 µg of total RNA was used in an oligo(dT) primed reaction in the presence of 200 µM aminoallyl-dUTP, 500 µM each dATP, dCTP, dGTP, 300 µM dTTP, 400 U of Superscript II polymerase (Invitrogen, Cergy Pontoise, France) in a final volume of 30 µl. After incubation at 42°C for 2 hours, RNA was hydrolyzed by adding 2 units of DNAse-free RNAse (Life Technologies) and incubation at 37°C for 15 minutes. The volume was adjusted to 500 µl with H20, aminoallyl-cDNA was purified by centrifugation in a microcon (YM-30 Millipore Amicon), eluted with 40 µl of TE and then dried in a vacuum. Samples were then resuspended in 10 µl sodium bicarbonate 0.1 M and mixed with monofunctional dye. Coupling reaction was performed during 30 minutes at room temperature in dark. The Cy3 and Cy5-labeled cDNAs were purified using a Microcon YM30, eluted with 100 µl of TE, mixed with 10 µg of human Cot-1 DNA, 10µg poly(A) and 10µg of yeast tRNA, then precipitated with 0.5 volume of 7.5 M sodium acetate (pH 5.2) in ethanol. The pellet was dissolved in 15 µl of hybridization solution (52% formamide, 1,75x denhardt’s, 0,35% SDS and 6x SSPE). The probe was heated for 2 min at 100°C, incubated at 37°C for 20-30 min and placed between slide and coverslip. The arrays were incubated overnight at 42°C in an humidified slide chamber (Corning) and washed in a first bath of 0.1x SSC with 0.1% SDS for 15 minutes followed by two washing steps in 0.1x SSC for 15 min each at room temperature. The arrays were dried by centrifugation at 600 rpm for 6 min. Fluorescence intensities of Cy3 and Cy5 were measured separately at 532 and 635 nm with a laser scanner (Axon Genepix4000B).
percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #1 (635 nm, Cy5)
F635 % Sat.
percentage of pixel saturation within feature
F532 Median
median pixel intensity of feature for Cy3 channel
F532 Mean
mean pixel intensity of feature for Cy3 channel
F532 SD
Cy3 pixel intensity standard deviation
B532 Median
median Cy3 feature background intensity
B532 Mean
mean Cy3 feature background intensity
B532 SD
Cy3 background pixel intensity standard deviation
% > B532+2SD
percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #2 (532 nm, Cy3)
F532 % Sat.
percentage of pixel saturation within feature
Ratio of Medians
ratio of the background subtracted median pixel intensity at the second wavelength (Cy3), to the background subtracted median pixel intensity at the first wavelength(Cy5)
Ratio of Means
ratio of the background subtracted median pixel intensity at the second wavelength (Cy3), to the background subtracted median pixel intensity at the first wavelength(Cy5)
VALUE
log ratio of PRE_VALUE
Mean of Ratios
arithmetic mean of the pixel-by-pixel ratios of the raw pixel intensities of wavelength 2 (Cy3) to wavelength 1 (Cy5)
PRE_VALUE
median of the pixel-by-pixel ratios of pixel intensities that have had the median background intensity subtracted of wavelength 2 (Cy3) to wavelength one (Cy5)
