|
| Status |
Public on Nov 30, 2012 |
| Title |
Patient2 Y31 |
| Sample type |
RNA |
| |
|
| Source name |
dermal mesenchymal stem cell
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: dermal mesenchymal stem cell
age: 45 years
gender: female
pasi: 14.7
|
| Growth protocol |
The dermises were separated from the epidermis by dispase and monoplast suspension was obtained by pipetting. The MSCs were cultured by adherent assay. Cells of 3rd passage were enrolled in the microarry.
|
| 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 |
Cy3
|
| Label protocol |
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.
|
| |
|
| Hybridization protocol |
the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.16.0) (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.
|
| Scan protocol |
Scanning is performed with the Axon GenePix 4000B microarray scanner. GenePix pro V6.0 is used to read the raw intensity of the image.
|
| 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>=50 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. Hierarchical clustering was performed using MEV software (v4.6, TIGR).
|
| |
|
| Submission date |
Nov 29, 2012 |
| Last update date |
Nov 30, 2012 |
| Contact name |
Ruixia Hou |
| E-mail(s) |
hrx0205@163.com
|
| Organization name |
Taiyuan City Central Hospital
|
| Department |
Dermatology
|
| Street address |
1,Dong San Dao Xiang, Jiefang Road
|
| City |
Taiyuan |
| State/province |
Shanxi |
| ZIP/Postal code |
030009 |
| Country |
China |
| |
|
| Platform ID |
GPL11434 |
| Series (1) |
| GSE42633 |
microRNA expression profile of dermal MSCs derived from psoriasis patients and normal control |
|
