|
| Status |
Public on Dec 10, 2012 |
| Title |
kidney_IRI+Hemin_3 |
| Sample type |
RNA |
| |
|
| Source name |
kidney, mices submitted to IRI and previous treatment with Hemin, replicate 3
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
surgery procedure: IRI
protection maneuver against iri: Hemin
|
| Treatment protocol |
Mice were anesthetized with Ketamine-Xylazine, a midline incision was made and both renal pedicles were cross-clamped for 45 minutes. During the procedure, animals were kept well hydrated with saline and at a constant temperature (~37oC) through a heating pad device. Subsequently, microsurgery clamps were removed, the abdomen closed and animals placed in single cages, warmed by indirect light until completely recovered from anesthesia. Animals were kept under adjustable conditions until sacrifice, namely 6 hours after renal reperfusion. In order to induce heme-oxygenase 1 (Hmox1) expression, mice received Hemin (Frontier Scientific, Canada) i.p., 25 mg/kg, 24 hours prior to surgery.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from kidney samples using Trizol reagent (Life Technologies) and purified using RNeasy Spin Columns (Qiagen, USA). RNA quantity was determined using a Nanovue spectrophotometer (GE Healthcare, USA). The RNA quality was performed using a 2100 Bioanalyzer with an RNA 6000 Nano kit and Ladder (Agilent Technologies, USA), according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Microarray-Based Gene Expression Analysis - Quick Amp Labeling (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (Qiagen). RNA concentration and purity was determined by reading the absorbance at 260 and 280 nm on a spectrophotometer (NanoVue, GE Health Care, USA).
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes and hybridized to Agilent Whole Mice Genome Oligo Microarrays (G4122F) for 17 hours at 65°C in a rotating Agilent hybridization oven.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Bundle using one color scan setting for 1x44k array slides.
|
| Description |
Gene expression of kidney tissue extracted from a mice model of renal Ischemia-reperfusion injury (IRI) submitted to pre-treatment with the drug Hemin
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software version 9.5.3.1 (Agilent) using default parameters (protocol GE1-v5_95 Feb07 and Grid: 014868_D_F_20090416) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jul 20, 2012 |
| Last update date |
Dec 10, 2012 |
| Contact name |
Hatylas Azevedo |
| Organization name |
University of São Paulo
|
| Department |
Pediatrics
|
| Street address |
Doutor Arnaldo Avenue, 455
|
| City |
São Paulo |
| State/province |
Sao Paulo |
| ZIP/Postal code |
01246903 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE39548 |
Transcriptome Analysis of Renal Ischemia/Reperfusion Injury and its Modulation by Ischemic Pre-Conditioning or Hemin treatment |
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