|
| Status |
Public on Jul 01, 2014 |
| Title |
HepG2 cells_cacao proanthocyanidin extract_100mg/L_5h_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Human hepatocellular liver carcinoma (HepG2) cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line source: American Type Culture Collection (ATCC, LGC Promochen, HB8065)
treatment group: cacao proanthocyanidin extract_100mg/L_5h
|
| Treatment protocol |
HepG2 cells were treated with 50 mg/L of epigallocatechin gallate, 100 mg/L of grape seed proanthocyanidin extract or 100 mg/L of cacao proanthocyanidin extract dissolved in DMSO (final DMSO concentration in growth media, 0.1%) for 5 h.
|
| Growth protocol |
The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Lonza, BE12-614) supplemented with 10% fetal bovine serum (Lonza, BE-14-802-F), 0.1 mM nonessential amino acids (Sigma, M7145), 100 U/ml penicillin, 100 µg/ml streptomycin (Lonza, BE-17-602E), 2 mM glutamine (Lonza, BE-17-605E), and 25 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, Sigma, H3375) in a humidified incubator with 5% CO2 at 37 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the miRNeasy Mini Kit (QIAGEN) following the manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
The probes were designed as the reverse complements of all major mature miRNAs and the mature sequences. Additional nucleotides were added to the 5' end of each capture oligonucleotide as necessary for the enzymatic extension in the labeling procedure. The probes were synthesized with intra-array replicates to increase the statistical confidence and to compensate for potential positional effects. As a result, the raw data files contained 7 data points for each miRNA. The intensities of blank probes, which consisted only of one single T nucleotide, were used for background corrections. Spike-in controls were also performed for the labeling efficiency
|
| |
|
| Hybridization protocol |
RNA was suspended in febit's proprietary miRNA Hybridization Buffer (25 μl per array). Hybridization was performed automatically for 16 h at 42ºC using a Geniom RT®-Analyzer. In the next step, the biochip underwent a stringent wash. Following the labeling procedure, febit ran a microfluidic-based primer extension assay. This assay utilizes the bound miRNAs as a primer for an enzymatic elongation with labeled nucleotides. The elongation was performed with Klenow Fragments and biotinylated nucleotides at 37ºC for 15 minutes. Finally, the biochip was washed automatically.
|
| Scan protocol |
For maximum sensitivity, febit used biotin, which was detected with streptavidin-phycoerythrin (SAPE), in combination with febit's consecutive Signal Enhancement (CSE) procedure. The feature recognition (using Cy3 filter set) and signal calculation were performed automatically within milliseconds
|
| Description |
B1
total RNA was extracted together with small RNAs (microRNAs)
|
| Data processing |
Spatial effects on the chip were investigated and corrected.
Intensity value distribution of the raw data was analyzed and, if required, normalized using variance stabilizing normalization (VSN).
Analysis and normalization were performed using R/Bioconductor packages including limma and VSN for normalization
|
| |
|
| Submission date |
Jul 12, 2011 |
| Last update date |
Jul 01, 2014 |
| Contact name |
Anna Arola-Arnal |
| E-mail(s) |
anna.arola@urv.cat
|
| Organization name |
Universitat Rovira i Virgili
|
| Street address |
Campus Sescelades. C/Marcel.lí Domingo s/n
|
| City |
Tarragona |
| State/province |
Tarragona |
| ZIP/Postal code |
43007 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13886 |
| Series (1) |
| GSE30575 |
microRNA profiling of HepG2 cells: control vs treatment with cacao, grape seed proanthocyanidin extract or epigallocatechin gallate |
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