|
| Status |
Public on Jul 31, 2012 |
| Title |
GFP over-expression 4 days |
| Sample type |
RNA |
| |
|
| Source name |
GFP over-expression 4 days
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary human neonatal foreskin keratinocytes
transgene: GFP
time: 4 d
|
| Treatment protocol |
NHEK cells were cultivated in 6-well dishes to 30%-50% confluence and infected using 1/50 final volume of 100x concentrated lentivirus containing pLenti6/V5Dest expression vectors for GFP and KLF9, respectively and 8 µg/ml protamine sulfate (Sigma-Aldrich, Hamburg, Germany). Cells were selected for 24 hours after transduction using 10 µg/ml puromycin for 24h.
|
| Growth protocol |
Normal human epidermal neonatal foreskin keratinocytes of three donors (NHEK-pool) were purchased from Lonza (Rockland, ME), seeded in 75 cm2 plastic flasks and cultured in complete Keratinocyte Growth Medium 2 (KGM-2) with growth factor supplements (Lonza, Rockland, ME) and 1 mM CaCl2. Cells were cultivated at 37 °C, 7% CO2 and 95% relative humidity.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested and total RNA was isolated using the High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) according to manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
For the linear T7-based amplification step, 100 ng of all samples were used. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies)
|
| |
|
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 1.2-1.65 ug Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 hours, 65 °C) to Agilent Whole Human Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 1 min. The last washing step was performed with acetonitrile.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies).
|
| Description |
Gene expression after 4d GFP over-expression
|
| Data processing |
Raw data of the hybridized microarrays were normalized using the R-Project-Bioconductor package Linear models for microarray data (limma). Background-correction was performed using the normexp function and the between-array-normalization quantile. An offset of 20 was added to stabilize the background correction and subsequently signals were log2 transformed. Genes were considered to be expressed if the background subtracted signal was above 2.6 times the standard deviation of the background signal in at least 75% of the microarrays.
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| |
|
| Submission date |
Feb 22, 2012 |
| Last update date |
Jul 31, 2012 |
| Contact name |
Florian Spoerl |
| E-mail(s) |
[email protected]
|
| Organization name |
Beiersdorf AG
|
| Department |
Aged Skin
|
| Lab |
Dry Aged Skin
|
| Street address |
Unnastr. 48
|
| City |
Hamburg |
| ZIP/Postal code |
20245 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE36014 |
Global changes in gene expression in primary keratinocytes after KLF9 overexpression |
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