|
| Status |
Public on Dec 31, 2013 |
| Title |
13.B.1 |
| Sample type |
RNA |
| |
|
| Source name |
pig 13, border region, treated
|
| Organism |
Sus scrofa |
| Characteristics |
strain: domestic
individual: 13
tissue: Border region
treatment: yes
|
| Treatment protocol |
The pigs were randomized and received either secretome of apoptotic peripheral mononuclear cells or medium-placebo via percutaneous intramyocardial delivery. One month post treatment, cardiac magnet resonance with late enhancement protocol was performed, and the pigs were humanely euthanized.
|
| Growth protocol |
Domestic pigs were pre-treated with per os 250 mg aspirin and 300 mg clopidogrel one day before induction of closed-chest reperfused myocardial infarction. After 1 month post-AMI, the pogs were randomized. During the follow-up, a daily dose of 100 mg aspirin and 75 mg clopidogrel were administered.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue-samples for RNA extraction were obtained directly after sacrification of the animals from myocardium in the remote border-(injection)-zone- and infarct core region, and stored in RNAlater (Qiagen, Germany) at -20°C. Total RNA was isolated from tissue samples with RNeasy Microarray Tissue Mini Kit (Qiagen, Germany) after manufacturer´s protocol. RNA quality was checked on RNA Nano chips via Agilent 2100 Bioanalyzer platform (Agilent Technologies), all RNA samples used for expression analysis had an RNA Integrity Number (RIN) higher than 6 indicating sufficient quality.
|
| Label |
Cy3
|
| Label protocol |
100 ng of each total RNA sample was used for the linear T7-based amplification step. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 1.65 μg Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 hours, 65 °C) to Agilent Whole Sus scrofa Genome Oligo Microarrays 4x44K V2 using hybridization chamber and oven. Finally, the microarrays were washed with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated (37 °C) Agilent Gene Expression Wash Buffer 2 for 1 min.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies) and analyzed by the Agilent Feature Extraction Software (FES).
|
| Description |
Gene expression 1 month after treatment
|
| Data processing |
Raw hybridization values were loaded into R (GNU R) using the R-package limma (v3.14.4), the values were background-corrected with method ‘normexp’ adding an offset of 1 and finally quantile normalized and log2-transformed.
|
| |
|
| Submission date |
May 27, 2013 |
| Last update date |
Dec 31, 2013 |
| Contact name |
Mariann Gyongyosi |
| E-mail(s) |
[email protected]
|
| Organization name |
Medical University of Vienna
|
| Street address |
Wahringer Gurtel 18-20
|
| City |
Vienna |
| ZIP/Postal code |
A-1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL16571 |
| Series (1) |
| GSE47397 |
Gene expression profiling in porcine chronic myocardial ischemia treated with intramyocardial injections of secretome |
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