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| Status |
Public on Jan 01, 2016 |
| Title |
C57BL/6_right thigh skeletal muscle after ischemia 2h and reperfusion 1d rep3 |
| Sample type |
RNA |
| |
|
| Source name |
C57BL/6_right thigh skeletal muscle after ischemia 2h and reperfusion 1d
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: right thigh femoralis muscle
|
| Treatment protocol |
Isolated skeletal muscle ischemia-reperfusion injury (IRI): Mice were anesthetized with an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine, given as an intraperitoneal injection (0.01 ml/g body weight). After the induction of anesthesia, the mice were restrained in a supine position on a heating pad to maintain body temperature at 37°C. The quadriceps muscle perfused based on the femoral artery of the mouse was carefully isolated away from the femoral bone and the underlying adductor muscle group. In the ischemic group, ischemia was induced by placing a microvascular clamp carefully across the proximal site of vascular pedicle for 2 h and then the microvascular clamp was removed. Good vascular inflow and outflow through the pedicle was verified under direct magnified vision. In the sham-operated control group, the muscle was isolated without microvascular clamp being applied. The incision wound was closed with interrupted sutures (4-0 nylon) and the animals were allowed to awaken in the remaining periods of reperfusion.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction by RNeasy Protect Animal Blood Kit (QIAGEN Cat. no. 73224). RNA purity was checked by optical density of NanoDrop ND-1000 and agarose electrophoresis with the result of A260/A280≧1.8, A260/A230≧ 1.8, no gDNA contamination. RNA integrity was measured by Agilent RNA 6000 Nano Assay (RIN≧6).
|
| Label |
Cy5
|
| Label protocol |
Small RNA population were enriched by (Nanosep100k, Pall Corp.) from 2.5 µg of total RNA. NHS-CyDye (Cy5, Amershan) were direct labeled to Guanine (G) by chemical labeling method using ULS™ microRNA Labeling Kit (EA-038, Kreatech).
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| |
|
| Hybridization protocol |
The pre-hybridization of MRmiOA v1 arrays were rehydrated by 100% ethanol following with deionized water. The slides were pre-hybridized with 5x SSPE, 0.1% SDS and 1% BSA at 42℃ for 2 hour. After the pre-hybridization, Cy5-labeled small RNA was hybridize on MRmiOA v1 in the presentation of the Phalanx OneArray hybridization buffer.
|
| Scan protocol |
The arrays were scanned by Axon GenePix 4000B scanner (635nm power 100 PMT 560-600 ; 532nm power 10, PMT 460) and quantify the fluoresence intensity.
|
| Description |
isc2h re1d-6
pure isolated skeletal muscle based on vascular pedicle
|
| Data processing |
The data processing was processed by R program (2.12.1). Import (Foreground minus background) value of each miRNA probes in triplicates from GPR files, plus the absolute value of minimum value of array to replace the week signal. Filter out probes that flag <0 in all samples. Porbes that passed the criteria were normalized by 75% media scaling between arrays.
The Matrix value is normalized intensities. Based on the normalized intensities, we calculate average ratios and find significant values for each gene by pairwise comparision (Ranking and hypothesis testing).
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| |
|
| Submission date |
Jun 07, 2013 |
| Last update date |
Jan 01, 2016 |
| Contact name |
Ching-Hua Hsieh |
| E-mail(s) |
m93chinghua@gmail.com
|
| Organization name |
Kaohsiung Chang Gung Memorial Hospital
|
| Department |
Plastic and Reconstructive Surgery
|
| Street address |
123, Ta-Pei road, Naio-Sung Hsiang
|
| City |
Kaohsiung Hsien |
| State/province |
Kaohsiung |
| ZIP/Postal code |
833 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL17252 |
| Series (1) |
| GSE47730 |
TLR4/NF-κB-responsive microRNAs in a mouse model of skeletal muscle ischemia-reperfusion injury |
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