|
| Status |
Public on Jul 06, 2015 |
| Title |
kidney_G01_Control_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
kidney
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Wister
Sex: male
weight: 200-300g
group: Group 1
injury: No
treatment: Control
time (h): 0
tissue: kidney
|
| Treatment protocol |
Briefly, rats were anesthetized by isofluorane inhalation induction followed by ketamine (40-80 mg/kg) and xylazine (5-10 mg/kg) intraperitoneally. Rats were premedicated with Valproic acid (150mg/kg), dexamethasone (3mg/kg) or saline (control) intraperitoneally thirty minutes prior to surgery. A sterile surgical site was prepared and midline incision was performed to access the abdominal cavity. Left kidney was located and its renal artery was clamped using an atraumatic microaneurysm clamp. The abdomen was reapproximated to prevent dehydration and maintain body temperature. Following 45 minutes of warm ischemia the abdomen was reopened and the renal clamp was removed to allow reperfusion. Reperfusion was confirmed by observing the color change in the kidney (pale to bright red). This was immediately followed by a right nephrectomy. The abdomen was then closed by suturing muscles with 6-O prolene and skin with 4-O nylon and clips.
|
| Growth protocol |
Male Wister rats (200-300g) were purchased from Harlan Laboratories and housed in accordance with the Guidelines for the Care and Use of Laboratory Animals. All experiments were conducted according to NIH and institutional guidelines with the approval of Madigan Army Medical Center Institutional Animal Care and Use Committee.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the kidney tissue using Trizol (Invitrogen, Carlsbad, CA) followed by column purification with miRNeasy-96 kits (Qiagen, GmbH, Germany) to remove residual salt and organic solvents. Total RNA quality and quantity were evaluated using an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA) and verified using the NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE).
|
| Label |
biotin
|
| Label protocol |
An initial input of 100 ng total RNA per sample was synthesized to cRNA using the Ambion Whole Transcript (WT) Expression Kit (Ambion, #4411974) according to manufacturer’s recommendations. Then 10 µg cRNA was used as a template to synthesize cDNA which were fragmented and labeled using the GeneChip WT Terminal Labeling Kit (Affymetrix, #901524).
|
| |
|
| Hybridization protocol |
Fragmented, labeled cDNA were hybridized to Affymetrix RatGene 1.1 ST array plates on an Affymetrix GeneTitan following manufacturer’s instructions using the GeneTitan Hybridization, Wash and Stain Kit for WT array plates (Affymetrix, #901622).
|
| Scan protocol |
Array plates were scanned immediately after hybridization, wash and staining by the Affymetrix GeneTitan using default settings.
|
| Data processing |
Microarray data was processed for background adjustment, normalization, and summarization using the interPLIER gene level method (background = PM-GCBG, normalization method = Sketch-Quantile) in Affymetrix Expression Console software (Version 1.1).
|
| |
|
| Submission date |
Jun 12, 2014 |
| Last update date |
Jul 07, 2015 |
| Contact name |
Christine Baer |
| E-mail(s) |
christine.e.baer2.ctr@mail.mil
|
| Organization name |
Excet, Inc. / USACEHR
|
| Department |
Environmental Health Program
|
| Street address |
568 Doughten Drive
|
| City |
Ft. Detrick |
| State/province |
MD |
| ZIP/Postal code |
21702-5010 |
| Country |
USA |
| |
|
| Platform ID |
GPL11534 |
| Series (1) |
| GSE58438 |
Renoprotective Effects of Valproic Acid and Dexamethasone in Acute Kidney Ischemia-Reperfusion Injury |
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