|
| Status |
Public on Jul 31, 2012 |
| Title |
Time point 2 (7.30 pm) subject 18 |
| Sample type |
RNA |
| |
|
| Source name |
Suction blister epidermis
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Epidermis
time point: Time point 2 (7.30 pm)
|
| Treatment protocol |
No treatment was performed. Epidermal suction blister samples were harvested at the indicated time points and instantly snap frozen in liquid nitrogen.
|
| Growth protocol |
-
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed using the Trizol method according to standard protol.
|
| 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 at 7.30 pm
|
| 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.
|
| |
|
| Submission date |
Feb 08, 2012 |
| Last update date |
Jul 31, 2012 |
| Contact name |
Florian Spoerl |
| E-mail(s) |
Florian.Spoerl@gmx.de
|
| 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) |
| GSE35635 |
Detection of circadian gene expression in human epidermal suction blister samples |
|
