|
| Status |
Public on Nov 18, 2015 |
| Title |
Sham |
| Sample type |
RNA |
| |
|
| Source name |
kidney tissue of Sham-operated mice
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57B/6
gender: male
tissue: kidney
|
| Growth protocol |
Healthy male C57B/6 mice (20-25 g) were maintained under the standard animal room conditions (temperature 21±1 °C; humidity 55–60%) with food and water ad libitum for one week before in vivo experiment. Animals were suffered renal bilateral ischemia reperfusion injury (IRI). Warm ischemic time was 30 min. Sham-operated mice, which underwent the same surgical procedure without placement of the vascular clamp served as controls (Sham group). Mice were sacrificed at 6, 12, 24 and 48 hours after reperfusion (n = 8 per time point).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Twenty mg of each renal tissue which had been stored in liquid nitrogen was obtained to extract RNA. Total RNA was extracted from each sample using Trizol reagent (Invitrogen, Shanghai, China).
|
| Label |
Cy3
|
| Label protocol |
Total RNA of 0.1 μg were dephosphorylated, denaturated and then labeled with Cyanine-3-CTP (Cy3) using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions,then followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1.
|
| |
|
| Hybridization protocol |
Cy3-labelled cRNA of 0.6 μg (specific activity >10.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 μl containing 25x Agilent fragmentation buffer and 10x Agient blocking agent following the manufacturers' instructions. On completion of the fragmentation reaction, 25 μl of 2x GEx Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent-070156 Human miRNA Microarray V19.0 8x60K(Agilent) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 μm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities as the raw data. Raw data were normalized in quantile algorithm with Genespring 12.0 (Agilent). Probe that at least 100.0 percent of samples in any 1 condition out of 2 conditions have flags in Detected were maintained.
|
| |
|
| Submission date |
Nov 17, 2015 |
| Last update date |
Nov 23, 2015 |
| Contact name |
Wenliang Zhu |
| E-mail(s) |
[email protected]
|
| Phone |
+86-451 8660 5353
|
| Organization name |
The Second Affiliated Hospital of Harbin Medical University
|
| Department |
1Institute of Clinical Pharmacology
|
| Street address |
Xuefu road 194
|
| City |
Harbin |
| State/province |
Heilongjiang |
| ZIP/Postal code |
150081 |
| Country |
China |
| |
|
| Platform ID |
GPL17912 |
| Series (1) |
| GSE75076 |
Global miRNA expression is temporally correlated with acute kidney injury in mice |
|
