|
| Status |
Public on Sep 29, 2015 |
| Title |
Stress-F1 (3) |
| Sample type |
RNA |
| |
|
| Source name |
Liver_stress
|
| Organism |
Mus musculus |
| Characteristics |
tissue: liver
strain: C57BL/6
gender: Male
age: 12 weeks
|
| Treatment protocol |
All mice were housed at 21±1°C with a humidity of 55±10% and a 12-hour light–dark cycle.
|
| Growth protocol |
C57BL/6 male mice aged 8 weeks were randomly assigned to control or stress group. Control mice were left undistributed and allowed contact with each other, while stressed mice were individually subjected to 2 hr/day of immobilization stress in 50-mL conical centrifuge tube (Corning Life Sciences, Tewksbury MA, USA) for 2 weeks (between 11:00 AM and 13:00 PM). After 2-week restraint stress, mice were placed with females for 2 days. Then, males were removed, and pregnant females were left alone with a shepherd shack until their offspring were 3 weeks of age. Finally, male offspring aged 12-week were sacrificed and total RNAs were extracted from hepatic tissues.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer’s instructions.
|
| Label |
Hy3TM fluorescent
|
| Label protocol |
One microgram of each sample was 3'-end-labeled with Hy3TM fluorescent label, using T4 RNA ligase by the following procedure: RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3TM), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C.
|
| |
|
| Hybridization protocol |
After stopping the labeling procedure, the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon) according to array manual. The total 25 μL mixture from Hy3TM-labeled samples with 25 μL hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min and then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization Systems (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA)
|
| Scan protocol |
Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon), and finally dried by centrifugation for 5 min at 400 rpm. Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).
|
| Data processing |
Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities>=30 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. After normalization, significant differentially expressed miRNAs were identified through Volcano Plot filtering. The file "normalized_data.txt" containing the normalized signal is available on the series record.
|
| |
|
| Submission date |
Sep 28, 2015 |
| Last update date |
Sep 29, 2015 |
| Contact name |
Yan Lu |
| E-mail(s) |
lu.yan2@zs-hospital.sh.cn
|
| Phone |
+86-21-64041990
|
| Organization name |
Zhongshan Hospital, Fudan University
|
| Department |
Department of Endocrinology and Metabolism
|
| Lab |
Zhongshan
|
| Street address |
180 Fenglin Road
|
| City |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
| |
|
| Platform ID |
GPL19128 |
| Series (1) |
| GSE73530 |
MicroRNA expression analysis of livers of F1 male offspring fathered from control mice or stressed mice |
|
