gender: male tissue: skin cell type: human dermal fibroblasts (HDF) stimulated with: normal human keratinocytes (NHK)-conditioned medium time point: 3h after stimulation
Treatment protocol
Conditioned media from normal human keratinocytes (NHK) were used to stimulate cells. Sub-confluent monolayers of fibroblasts were grown in individual flasks, and conditioned medium (0.2-0.3 mL/cm2) was collected as follows: i. NHK conditioned medium (K): KSFM (supplemented with rhEGF and BPE) was added to keratinocyte culture flask and collected after 16h. The After each conditioning treatment the collected media were centrifuged (200 xg/ 5 min) and then was used to stimulate other cells or tested. Time and volume was considered to have adequate soluble factors content and sufficient medium nutrients. RNA was collected fro expression profiling after 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 18 and 24h of treatment.
Growth protocol
Human dermal fibroblasts (HDF) were derived from foreskin as previously described (König & Bruckner-Tuderman, 1994; Stark et al, 1999). HDF obtained from the outgrowth of explants culture and were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% L-glutamate, penicillin G and streptomycin (PAN-Biotech, Aidenbach, Germany). Cells were kept under a humidified environment with 5% CO2 and 3oC. Fibroblasts at passages 2-8 and a density of 5-8 x 10^4 cells/cm^2 were used in all experiments.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.
Label
Cy3
Label protocol
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
Hybridization protocol
Hybridization was performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes. After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals were developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays were dried and scanned.
Scan protocol
Microarray scanning was done using a Beadstation array scanner, setting adjusted to a scaling factor of 1 and PMT settings at 430. Data extraction was done for all beads individually
Description
Array ID: 4675283074
Data processing
All arrays were normalized together using the quantile normalization algorithm without background subtraction (Illumina BeadStudio Software).
