|
| Status |
Public on Jun 25, 2013 |
| Title |
endothelial cell-H1 [miRNA] |
| Sample type |
RNA |
| |
|
| Source name |
endothelial cell-H1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: endothelial cells
|
| Growth protocol |
Human umbilical vein endothelial cells and human dermal fibroblasts cells were were cultured in DMEM/F12 medium supplemented with 10% FBS.Adipose tissues obtained from the patients should be washed 3 times by PBS with 1% penicillin/streptomycin and then carefully minced by sterile operation scissors. The minced tissues were then enzymatically dissociated for 45 minutes at 37 °C by 0.15% collagenase type I (GIBCO). Then the suspension was then neutralized with isometric culture medium and centrifuged at 500g for 5 minutes. The cell pellet was resuspended in DMEM/F12 medium (GIBCO) supplemented with 10% FBS (GIBCO) or 10% KSR (Invitrogen)at a density of 2x106 cell/mL. Cell cultures were maintained at 37 °C in a humidified incubator supplemented with 5% CO2. Passage 3 cells were used for whole human genome microarray.
|
| 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 |
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 5th generation Array (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.
|
| Description |
microRNA expression of human endothelial cells
SAMPLE 8
|
| Data processing |
The intensity of green signal is calculated after background subtraction and four Replicated spots of each probe on the same slide have been averaged. We use Median Normalization Method to obtain 'Normalized Data', Normalized Data= (Foreground-Background)/median, the median is 50 percent quantile of microRNA intensity which is larger than 50 in all samples after background correction. After normalization, the statistical significance of differentially expressed miRNA was analyzed by T-test.
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| |
|
| Submission date |
Jun 24, 2013 |
| Last update date |
Jun 25, 2013 |
| Contact name |
Min Li |
| E-mail(s) |
minlicq@gmail.com
|
| Organization name |
Tongji University
|
| Department |
Life Sciences and Technology
|
| Street address |
50 Chifeng Rd., Yangpu District
|
| City |
Shanghai |
| ZIP/Postal code |
200092 |
| Country |
China |
| |
|
| Platform ID |
GPL14765 |
| Series (2) |
| GSE48227 |
Differential expression of microRNAs in adipose stem cells |
| GSE48228 |
Differential expression in adipose derived stem cells |
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