|
| Status |
Public on Jul 30, 2012 |
| Title |
Wound tissue day21 vs. day0 uninjured skin rep.2 (dye flip) high int. scan |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D0 skin
|
| Organism |
Sus scrofa |
| Characteristics |
scan method: high int. scan
tissue: skin
sample day: D0
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted from tissue stabilized in RNALatter using QIAGEN RNeasy Lipid Tissue Mini kit and automatic isolator QIAcube according to supplier standard protocol and instructions.
|
| Label |
Cy5
|
| Label protocol |
0.5 µg of total RNA was amplified labeled with Cy3 and Cy5-CTPs using Low Input QuickAmp Labeling Kit (Agilent) according to manufactuers's instructions. In two animals, skin samples were labeled Cy3 and wound sample Cy5, in other two animals, samples were labeled inversly using flip dye.
|
| |
|
| Channel 2 |
| Source name |
D21 wound tissue
|
| Organism |
Sus scrofa |
| Characteristics |
scan method: high int. scan
tissue: wound scar tissue
sample day: D21
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted from tissue stabilized in RNALatter using QIAGEN RNeasy Lipid Tissue Mini kit and automatic isolator QIAcube according to supplier standard protocol and instructions.
|
| Label |
Cy3
|
| Label protocol |
0.5 µg of total RNA was amplified labeled with Cy3 and Cy5-CTPs using Low Input QuickAmp Labeling Kit (Agilent) according to manufactuers's instructions. In two animals, skin samples were labeled Cy3 and wound sample Cy5, in other two animals, samples were labeled inversly using flip dye.
|
| |
|
| |
| Hybridization protocol |
Samples from skin and wound from the same animal, labeled with opposite color were hybridized. After 30 min/60 C fragmentation of samples with fragmentation buffer and blocking agent (Agilent), Hi-RPM Hybridization buffer (Agilent) was added and mixed. Mixture was applied to microarrays and enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential.
Hybridization was performed at 65°C for 17h. Slide were washed thoroughly using GE Wash Buffer 1 at RT, Wash Buffer 2 at 37°C and HPLC acetonitril (Sigma), followed by stabilization using Stabilization and drying solution (Agilent)
|
| Scan protocol |
Slides were scanned using Molecular Device Axon Gene Pix 4000B (ver. 2010) at 5 um resolution, with 3 passages at low PMT settings (L-samples) and 3 passages high PMT settings (H-samples), in order to detect wide range of expressions.
|
| Description |
Biological replicate 2 of 4. Wound tissue day 21 vs day 0 uninjured skin. High intensity scan. Scan 2 of 2.
|
| Data processing |
Gene Pix Pro 7.2 was used for image analysis and bad spots removal (low intensity, saturated, control array spots). Acuity 4.1 was used for data compilation, LOWESS normalization and data analysis. Where applicable, flip dye function was applied on flip dye data (logR => -logR). Values, in the Sample tables, are not merged (high and low intensity scan). Positive values signify increase of transcript in wound sample in comparison to skin. Negative values signify decrease in skin. Hi and Low intensity scans data were merged using average of LOWESS logR, for further analysis.
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| |
|
| Submission date |
May 11, 2012 |
| Last update date |
Jul 30, 2012 |
| Contact name |
Rastislav Slavkovský |
| E-mail(s) |
slavkovsky@contipro.cz
|
| Phone |
+420732741171
|
| Organization name |
Contipro Biotech
|
| Department |
R&D
|
| Lab |
lab. of cell physiology and biofilm
|
| Street address |
Dolni Dobrouc 401
|
| City |
Dolni Dobrouc |
| ZIP/Postal code |
561 02 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL15561 |
| Series (1) |
| GSE37947 |
Transcriptome profiling during cutaneous porcine wound repair. |
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