|
| Status |
Public on Dec 15, 2023 |
| Title |
Intestine_10Gy_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Intestine
|
| Organism |
Mus musculus |
| Characteristics |
gender: male
age: 8 weeks
|
| Treatment protocol |
Eight-week-old mice were exposed to X-rays (MBR-1520R-3 X-ray machine, Hitachi Medical Corporation) at a dose rate of 1.0 Gy/min (150 kVp, 20 mA, 0.5 mm aluminum and 0.3 mm copper filters). Blood obtained from the heart was collected in BD Microtainer tubes with serum separator additive BD Microgard closure (Becton Dickinson Biosciences). Blood was then centrifuged at 6000 × G at room temperature for 3 min, and serum was collected. Feces were directly collected in tubes. Small intestines were collected and stored at -80°C before use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs from the small intestine, serum, and feces were extracted using the Isogen II reagent and ethachinmate (Nippongene,) according to the manufacturer’s instructions. RNA concentration from the small intestine was assessed using the NanoDrop spectrophotometer (NanoDrop Technologies). RNA samples had 260/280 nm absorbance ratios of 1.8–2.0. RNA concentration of serum and feces was measured using a Quant-iT RiboGreen RNA Reagent and kit (Thermo Fisher Scientific) and Fluoroskan Ascent (Thermo Fisher Scientific) according to manufacturer’s instructions. The quality of total RNAs was confirmed using Agilent 2100 Bioanalyzer and Agilent RNA 6000 Pico kit (both Agilent Technologies), according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine 3(Cy3)-labeled miRNAs were synthesized from total RNA of irradiated (10 Gy) and non-irradiated (0 Gy) samples (small intestine, serum and feces) using a miRNA Complete Labeling Reagent and Hyb kit (Agilent Technologies)., according to the manufacturer’s instructions.
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| |
|
| Hybridization protocol |
SurePrint G3 mouse miRNA microarray slides (8 × 60 K, Ver.21.0) were hybridized with the Cy3-labeled miRNA in a hybridization solution prepared with a Gene Expression Hybridization Kit (Agilent Technologies) for 20 hours at 55°C in a rotating hybridization oven (Agilent Technologies), according to the manufacturer’s instructions. After hybridization, microarray slides were washed for 5 min at room temperature with GE Wash Buffer 1 (Agilent Technologies) and for 5 min at 37°C with GE Wash buffer 2 (Agilent Technologies), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the SureScan Microarray Scanner (G4900DA) (Agilent Technologies) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 2 μm high sensitivity, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
exposed to 10 Gy of X-rays
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5 (Agilent Technologies) using default parameters (Protocol name: miRNA_1105_Oct12 and Grid name: 070155_D_F_20141006) to obtain background subtracted and spatially detrended Processed Signal intensities. The expression data obtained were processed using the GeneSpring GX14.5 software (Agilent Technologies) to normalize all values to 90% percentile shift on the respective microarrays, followed by normalization of the median expression level of all samples. The increase and decrease of more than 2.0-fold miRNAs at 10 Gy against 0 Gy were selected.
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| |
|
| Submission date |
Nov 15, 2023 |
| Last update date |
Dec 15, 2023 |
| Contact name |
Mitsuru Chiba |
| E-mail(s) |
mchiba32@hirosaki-u.ac.jp
|
| Phone |
+81172395965
|
| Organization name |
Hirosaki University
|
| Department |
Graduate School of Health Sciences
|
| Lab |
Department of Bioscience and Laboratory Medicine
|
| Street address |
66-1 Hon-cho
|
| City |
Hirosaki |
| State/province |
Aomori |
| ZIP/Postal code |
036-8564 |
| Country |
Japan |
| |
|
| Platform ID |
GPL22383 |
| Series (1) |
| GSE247876 |
Extracellular miRNAs in serum and feces of mice exposed to high-dose radiation |
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