|
| Status |
Public on Mar 10, 2003 |
| Title |
ACG010 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HaCaT keratinocytes day3 proliferating
|
| Organism |
Homo sapiens |
| Extracted molecule |
total RNA |
| |
|
| Channel 2 |
| Source name |
HaCaT keratinocytes day10 cell density differentiated
|
| Organism |
Homo sapiens |
| Extracted molecule |
total RNA |
| |
|
| |
| Description |
The keratinocyte cell line HaCaT culture was performed in DMEM (Invitrogen), 10% FCS (Hyclone) at 37°C and 5% CO2 incubator. Cells were seeded at 10 000 cells/cm2 and cultured for three days (proliferation) or for ten days (differentiation). Medium was changed every two days.
Total RNA was isolated using the Trizol reagent (Invitrogen) and dissolved in RNAse-free water. RNA quality was checked by 1 % agarose gel electrophoresis and concentration was measured by absorbance at 260 nm.
Target cDNA was generated by direct incorporation of fluorescent nucleotide analogues during reverse transcription. For each labelling experiment, 20 µg of total RNA was submitted to an oligo(dT) primed reaction in the presence of 60 µM Cy3 or Cy5-dUTP (Amersham Biosciences), 500 µM each dATP, dCTP, dGTP, 200 µM dTTP, 4 µg oligo(dT) and 400 U of Superscript II (Invitrogen) in a final volume of 30 µl. After incubation at 42°C for 2 hours in a dark room, RNA was treated with 2 units of DNAse-free RNAse (Invitrogen) at 37°C for 15 min. The volume was adjusted to 500 µl with TE (10 mM TrisHCl pH 7.4, 1 mM EDTA) and non-incorporated nucleotides were separated from labelled cDNA by centrifugation in a YM-30 Microcon filter (Millipore Amicon). The volume of each labelled cDNA was then adjusted to 40 µl.
Cy3 and Cy5-labeled cDNAs were subsequently mixed with 10 µg of human Cot-1 DNA (Invitrogen), 10µg poly(A) (Sigma-Aldrich) and 10µg of yeast tRNA (Invitrogen) 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 hybridisation solution (50% 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.
Florescence intensities of Cy3 (532 nm) and Cy5 (635 nm) were measured separately with a laser scanner (Axon Genepix4000B).
|
| |
|
| Submission date |
Mar 10, 2003 |
| Last update date |
Oct 28, 2005 |
| Contact name |
Jerome Lamartine |
| E-mail(s) |
lamartin@dsvidf.cea.fr
|
| Phone |
33 1 60 87 34 84
|
| Fax |
33 1 60 87 34 98
|
| Organization name |
CEA
|
| Department |
Departement de Radiobiologie et Radiopathologie
|
| Lab |
Service de Genomique Fonctionnelle
|
| Street address |
2 rue Gaston Cremieux
|
| City |
Evry |
| ZIP/Postal code |
F91057 |
| Country |
France |
| |
|
| Platform ID |
GPL263 |
| Series (1) |
| GSE336 |
HaCaT keratinocytes day3 vs HaCaT keratinocytes day10 |
|
| Data table header descriptions |
| ID_REF |
|
| F635 Median |
median pixel intensity of feature for Cy5 channel |
| F635 Mean |
mean pixel intensity of feature for Cy5 channel |
| F635 SD |
Cy5 pixel intensity standard deviation |
| B635 Median |
median Cy5 feature background intensity |
| B635 Mean |
mean Cy5 feature background intensity |
| B635 SD |
Cy5 background pixel intensity standard deviation |
| % > B635+2SD |
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) |
