|
| Status |
Public on Feb 11, 2016 |
| Title |
DP4-a |
| Sample type |
RNA |
| |
|
| Source name |
cultured dermal papilla cell
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: scalp specimens
cell type: DP
passage: Early-passage (4)
|
| Growth protocol |
The DP was cultured in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal serum at 37°C in a humidified atmosphere containing 5% CO2. DP cells were cultivated continuously
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) and an miRNeasy mini kit (QIAGEN) according to manufacturer’s instructions. RNA quality and quantity was measured by using a Nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA integrity was determined by gel electrophoresis.
|
| Label |
Hy3
|
| Label protocol |
The samples were labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit(Exiqon, Vedbaek, Denmark).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 samples were hybridized on the miRCURY™ 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 |
Following the washing steps the slides were scanned using the Axon GenePix 4000B microarray scanner.Scanned images were imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction
|
| Description |
we used passage-4 DP cells only
|
| Data processing |
After low intensity miRNAs filtering, raw signal intensities are normalized in Median method. (miRNAs that intensities>=30 in all samples are chosen for calculating normalization factor)
Expressed data were normalized using the median. After normalization, differentially expressed miRNAs were identified through Volcano Plot filtering. Hierarchical clustering was performed using MEV software (v4.6, TIGR). The threshold value we used to screen up- and down-regulated miRNAs was a fold change >=1.50 and a P-value <=0.05.
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| |
|
| Submission date |
Feb 11, 2016 |
| Last update date |
Feb 11, 2016 |
| Contact name |
Yang Liu |
| E-mail(s) |
yangsweety@aliyun.com
|
| Phone |
15829781809
|
| Organization name |
Shantou University Medical College
|
| Department |
Department of Histology and Embryology
|
| Lab |
Histology and Embryology
|
| Street address |
No.22, Xingling Street, Jinping District
|
| City |
Ghantou |
| State/province |
Guangdong |
| ZIP/Postal code |
515000 |
| Country |
China |
| |
|
| Platform ID |
GPL21399 |
| Series (1) |
| GSE77825 |
miR-195-5p Regulates Hair Follicle Inductivity of Dermal Papilla Cells by Suppressing Wnt/β-catenin Activation |
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