|
| Status |
Public on Sep 04, 2014 |
| Title |
Ischemia_Reperfusion_24h_1 |
| Sample type |
RNA |
| |
|
| Source name |
Retina_Experimental sample_24h
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Long Evan’s hooded rat
gender: male
sample group: Ischemia reperfusion
time point: 24hr post-ischemia
tissue: whole retina
|
| Treatment protocol |
To induce ischemia, rats were first anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Tetracaine hydrochloride 0.5% and tropicamide 1% ophthalmic drops were administered to provide local anesthesia and mydriasis, respectively. For induction of high intra-ocular pressure (IOP) the anterior chamber of one eye was cannulated with a 30 gauge needle attached to an elevated 1L sterile saline reservoir. High IOP of 110 mmHg was maintained for 60 min. Reperfusion proceeded for the 5 time points: 0h, 2h, 24h, 48h and 7 days after which animals were euthanized and tissues were collected. The same procedures were administered to rats receiving sham surgeries except that high IOP was not induced. IOP was measured in mmHg at 15, 30 and 60min using a TonoLab tonometer device (Colonial Medical Supply, Franconia, NH). Retinas were dissected from each eye, flash frozen in liquid nitrogen, and stored in -80°C for subsequent use.
|
| Growth protocol |
Long Evan’s hooded male rats (Harlan, Indianapolis, IN) weighing 250 to 300g were used. All animals were housed on a 12-hour light– dark cycle with food and water. The treatment and care of all animals used in this study were approved by the University of Louisville Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with the ARVO Statement for the Use and Care of Animals in Ophthalmic and Vision Research.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Retinas were homogenized in lysis/binding buffer (Ambion, Inc.) using a motorized rotor-stator homogenizer. Total RNA was isolated using mirVana™ miRNA Isolation Kit (Ambion, Inc, Austin TX). The quality of the RNA was checked with the aid of the spectrophotometer (NanoDrop Technologies, Wilmington, DE). The RNA integrity numbers (RIN) for all samples were greater than 8.
|
| Label |
Cy3
|
| Label protocol |
100ng total RNA was used to generate fluorescent miRNA with the aid of Calf Intestine Alkaline Phosphatase (CIP) kit (GE Healthcare, Ca E2250Y), T4 RNA Ligase kit (GE Healthcare, CA E2050Y), Agilent’s miRNA Labeling Reagent and Hybridization kit provided with the Cyanine 3-pCP (Agilent, Wilmington De 5190-0408). Briefly, the RNA was treated with CIP (16U/uL) and denatured with 100% DMSO. The dephosphorylated RNA was ligated to pCP-Cy3 by T4-RNA Ligase. The labeled RNA was purified with the Micro Bio-Spin 6 Column (Bio-Rad, CA 732-6221). The desalted labeled RNA was dried down using a Speed-vac.
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| |
|
| Hybridization protocol |
The dried RNA sample was re-suspended in 18μl of DNase/RNase-free water and then hybridized to Agilent rat miRNA microarrays (8x15K feature with 350 miRNA, CA G4472A) with hybridization buffer at 550C for 20 hours. After hybridization, the microarray slides were washed with Agilent gene expression wash buffer kit (Agilent, 5188-5327): buffer 1 at room temperature for 5min; and buffer 2 at 370C for 5min.
|
| Scan protocol |
The microarray slides were scanned with the aid of an Agilent microarray scanner (G2565BA) with a setting for one-color using the green channel and 5um resolution. The one-color microarray images (.tif) were extracted with the aid of Feature Extraction software (v 9.5.1, Agilent).
|
| Description |
IR-25-24h.txt
|
| Data processing |
The raw data files with total 350 miRNAs extracted from Agilent Feature extraction software were further processed and analyzed by GeneSpring GX10.0 software. The raw data were at first normalized with the following conditions and then filtered by the flag using GeneSpring GX10.0 software. The normalization included log2 transformation, per chip normalization to 75% quantile and dropped per gene normalization to median. Filtered on the flag included miRNAs where at least 100 percent of samples in any 1 out of 15 conditions have flags in Present or Marginal. We retained the 219 normalized miRNAs for the further statistical analysis.
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| |
|
| Submission date |
Sep 03, 2014 |
| Last update date |
Sep 04, 2014 |
| Contact name |
Nigel G. F. Cooper |
| E-mail(s) |
nigelcooper@louisville.edu
|
| Organization name |
University of Louisville
|
| Department |
Anatomical Science and Neurobiology
|
| Street address |
500 S Preston St.
|
| City |
Louisville |
| State/province |
KY |
| ZIP/Postal code |
40292 |
| Country |
USA |
| |
|
| Platform ID |
GPL10906 |
| Series (1) |
| GSE61072 |
Time-Dependent miRNA Profiling Following Ischemia/Reperfusion Injury in Rat Retina |
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