|
| Status |
Public on Jun 11, 2014 |
| Title |
co_24h_445 |
| Sample type |
RNA |
| |
|
| Source name |
untreated primary human dermal fibroblasts, 24h, subject 10
|
| Organism |
Homo sapiens |
| Characteristics |
individual: subject 10
cell type: primary dermal fibroblasts
treatment: static
time: 24h
|
| Treatment protocol |
Mechanical stretching was performed on flexible silicon membranes using FX-4000T™ Tension Plus™ System (Flexercell International Corporation, Hillsborough, USA). Cells were seeded on Bioflex culture plates and after 24 h subjected to stretching experiments or left untreated as control. Cyclic stretch was applied for 5 h and 24 h in the FX-4000T ™ Tension Plus™ System with 16% Elongation, 0.5 Hz in a half sinus regimen. Cell alignment was observed at the outer circular region of the well. Control cells were left static for 5 h and 24 h, as well.
|
| Growth protocol |
Primary human dermal fibroblasts were isolated from skin biopsies obtained from plastic surgery by collagenase digestion and stored at -170°C in DMSO until usage. All donors provided written, informed consent. For experiments cells were cultured only until the third passage in Dulbecco’s modified Eagle’s Medium (DMEM, 4.5 g/l glucose) containing 10% fetal calf serum, 2 mM L-alanyl-L-glutamine and 0.1 mg/ml penicillin/streptomycin at 37°C in an atmosphere of 5% CO2 and 90% humidity.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To separate the inhomogeneous stretching areas of the BioFlex culture plates the silicon membranes were punched with a 2 cm diameter punch. Isolation of RNA was done with Rneasy Mini kit according to manufacturer product information from the outer cicular region of the well. DNA and RNA were precipitated with 70 % ethanol and bound to a column. DNA was digested using DNAse I. RNA was eluted with 30 µl RNAse free water and subjected to Experion automated electrophoresis using the Experion RNA StdSens Analysis Kit (Bio-Rad, Hercules, USA) for quality control.
|
| Label |
Cy3
|
| Label protocol |
For the linear T7-based amplification step, 100ng of each total RNA sample was 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 ND-1000 Spectrophotometer (NanoDrop Technologies)
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| |
|
| 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) (Design ID 028004). Briefly, 600 ng Cy3-labeled fragmented cRNA in hybridization buffer was hybridized 17 h at 65°C to Agilent whole genome oligo microarrays 8X60K 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 (G2505C, Agilent Technologies, Palo Alto, USA)..
|
| Description |
Gene expression after 24h cell culture
Reu_20
|
| Data processing |
The Agilent Feature Extraction Software (FES 10.7.3.1 ) was used to read out and process the microarray image files. The preprocessing proper started with the conversion of the data after using Agilent Feature Extraction software in a format suitable for all subsequent analysis steps, which were mainly performed with the R statistical software and its Bioconductor packages. For the preprocessing the agi4x44kpreprocess package was used. To this end an annotation package for the Agilent Whole Genome 8x60K chip has (previously) been created. After reading the data files into R, all data were background-corrected. Data were then normalized between arrays using quantile-normalization and transformed to log2-scale, which enabled comparison of samples loaded on different arrays. After normalization, a set of quality control steps were performed to filter low-quality probes. Filtering of probes was based on quality flags set by the Agilent Feature Extraction Software. Most of the probes (~42 %) were filtered because of not being sufficiently above background. Finally, the processed data were stored in formats that allowed further downstream analyses and easy access.
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| |
|
| Submission date |
Jun 11, 2014 |
| Last update date |
Jun 11, 2014 |
| Contact name |
Maria Reichenbach |
| Organization name |
Freie Universität Berlin
|
| Department |
Institute for Chemistry and Biochemistry
|
| Lab |
Signal transduction
|
| Street address |
Thielallee 63
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE58389 |
Cyclic mechanical stretch-induced gene expression |
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