|
| Status |
Public on Aug 31, 2010 |
| Title |
HealthyEpidermis_T4vsT1_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
T1 cell fraction (basal keratinocytes) [reference from sample 1]
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Female
health state: healthy
|
| Growth protocol |
Cells were not cultured (direct processing from ex vivo samples)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction with QIAGEN RNeasy mini or midi kits
|
| Label |
Cy3 on AQQ002 slide, Cy5 on swap-array AQQ003
|
| Label protocol |
Reverse transcription of 10-20µg of total RNA with Superscript II enzyme and oligo-dT12-18 primers (Invitrogen) in the presence of aminoallyl-dUTP (Sigma-Aldrich), purification with cleanup module of Labelstar Array kit (Qiagen), labelling with monofunctional NHS ester Cy3 or Cy5 dye (Amersham), purification, pooling of samples to compare and concentration under vacuum followed by addition of 50µl of hybridization buffer containing 2x SSC, 0.1% SDS and 0.1% salmon sperm DNA (Invitrogen)
|
| |
|
| Channel 2 |
| Source name |
T4 cell fraction (granular keratinocytes) [test from sample 1]
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Female
health state: healthy
|
| Growth protocol |
Cells were not cultured (direct processing from ex vivo samples)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction with QIAGEN RNeasy mini or midi kits
|
| Label |
Cy5 on AQQ002 slide, Cy3 on swap-array AQQ003
|
| Label protocol |
Reverse transcription of 10-20µg of total RNA with Superscript II enzyme and oligo-dT12-18 primers (Invitrogen) in the presence of aminoallyl-dUTP (Sigma-Aldrich), purification with cleanup module of Labelstar Array kit (Qiagen), labelling with monofunctional NHS ester Cy3 or Cy5 dye (Amersham), purification, pooling of samples to compare and concentration under vacuum followed by addition of 50µl of hybridization buffer containing 2x SSC, 0.1% SDS and 0.1% salmon sperm DNA (Invitrogen)
|
| |
|
| |
| Hybridization protocol |
cDNA were deposited on previously re-hydrated and blocked microarrays under coverslip, and slides were placed into hybridization chamber for 16 hours at 50°C. After 4 washes (2x SSC + 0.1% SDS ; 1x/0.2x/0.05x SCC) and drying, slides were scanned.
|
| Scan protocol |
Slides were scanned on a GenePix 4000A scanner and image intensity data were extracted with GenePix Pro 6.0 analysis software
|
| Description |
test (T4) and reference (T1) are obtained from the same biological sample
AQQ002 and AQQ003
|
| Data processing |
Median intensities of each spot, local background substraction, log transformation and lowess normalization using Bioplot software. Spots with signal intensity greater than average signal for all buffer spots plus 2 SD and detected by more than half of the samples (i.e. with 4 values at least) were analyzed, other were removed from analysis. Note that the array design includes for a very few genes (including negative controls and ACTB / GAPD housekeeping genes) multiple spot replicates; when several of the spot replicates were positive, corresponding ratios where merged, and by convention the ID mentioned in the Matrix corresponds to the ID of the first spot in the GPL1946 table registered in GEO. Note also that ID in .gpr raw data files do NOT correspond to ID in GPL1946 Table, but to the INTERNAL_ID.
|
| |
|
| Submission date |
Oct 17, 2008 |
| Last update date |
Nov 26, 2009 |
| Contact name |
Nicolas Mattiuzzo |
| E-mail(s) |
nicolas.mattiuzzo@gmail.com
|
| Phone |
+33 56158433
|
| Organization name |
Centre National de la Recherche Scientifique (CNRS, France)
|
| Department |
UMR CNRS-UPS (Univ. Paul Sabatier) 5165
|
| Lab |
Unité Différenciation Epidermique et Autoimmunité Rhumatoïde
|
| Street address |
CHU Purpan, place du Dr Baylac, TSA40031
|
| City |
Toulouse |
| ZIP/Postal code |
31073 |
| Country |
France |
| |
|
| Platform ID |
GPL1946 |
| Series (1) |
| GSE13263 |
Study of keratinocyte differentiation in normal human epidermis |
|
