|
| Status |
Public on May 01, 2014 |
| Title |
Treatment 4th group Replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
HepG2 human hepatocellular carcinoma cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2 (HB-8065)
cell passage: 3
cell type: human hepatocellular carcinoma cells
treatment: Triptolide 200 nM 24 h
|
| Growth protocol |
The cells were grown in Dulbecco’s Modified Eagle Medium (Gibco BRL) with 10% fetal bovine serum (Gibco BRL) and were maintained in an atmosphere of 5% CO2 in a humidified 37 °C incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Hy3
|
| 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.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. Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).
|
| Scan protocol |
Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).
|
| Description |
Biological replicate 2 of 3. HepG2 cells, treated with 200 nM triptolide for 24 h, harvested after several passages.
|
| 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, differentially expressed miRNAs were identified through Fold Change filtering. Hierarchical clustering was performed using MEV software (v4.6, TIGR).
|
| |
|
| Submission date |
Aug 10, 2012 |
| Last update date |
May 01, 2014 |
| Contact name |
Changquan Ling |
| E-mail(s) |
lsg54124@hotmail.com
|
| Phone |
86-21-81871551
|
| Organization name |
Second Military Medical University
|
| Department |
Department of Traditional Chinese Medicine
|
| Street address |
No. 800,Xiangyin Road
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200433 |
| Country |
China |
| |
|
| Platform ID |
GPL11434 |
| Series (1) |
| GSE40037 |
Triptolide alters the miRNA expression profiles in human hepatocellular carcinoma HepG2 cells |
|
