|
| Status |
Public on Sep 30, 2015 |
| Title |
S. japonicum infected pooled mice plasma Replicate 1 (I1) |
| Sample type |
RNA |
| |
|
| Source name |
S. japonicum infected pooled mice plasma
|
| Organism |
Mus musculus |
| Characteristics |
tissue: plasma
status: 25d post S. japonicum infection
Sex: Male
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer’s instructions, which efficiently recovered all RNA species, including miRNAs. RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel electrophoresis.
|
| Label |
Hy3
|
| Label protocol |
After RNA isolation from the samples, the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling. 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.
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| |
|
| 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), which provides an active mixing action and constant incubation temperature to improve hybridization uniformity and enhance signal. 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).
|
| Scan protocol |
the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).
|
| Description |
the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon) according to array manual
|
| 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, differentially expressed miRNAs were identified through Fold Change filtering. Hierarchical clustering was performed using MEV software (v4.6, TIGR).
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| |
|
| Submission date |
Nov 10, 2014 |
| Last update date |
Sep 30, 2015 |
| Contact name |
Lihui Zhu |
| E-mail(s) |
zlh309@163.com
|
| Organization name |
Shanghai Jiaotong University
|
| Department |
School of Agriculture and Biology
|
| Lab |
Shanghai Key Laboratory of Veterinary Biotechnology
|
| Street address |
800 Dongchuang Road
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200240 |
| Country |
China |
| |
|
| Platform ID |
GPL16016 |
| Series (1) |
| GSE63135 |
Altered levels of circulating miRNAs are associated with Schistosoma japonicum infection in mice |
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