|
| Status |
Public on Aug 07, 2012 |
| Title |
bap-1-48h-Ex2 _12X |
| Sample type |
RNA |
| |
|
| Source name |
HepG2 cells (ATCC: HB-8065)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: hepatocellular carcinoma
cell line: HepG2 cells (ATCC: HB-8065)
treatment: bap
time: 48h
|
| Treatment protocol |
HepG2 cells were exposed to 2 μM of BaP and to a vehicle control (0.5% DMSO) during 6, 12, 24 and 48 hours in three independent experiments.
|
| Growth protocol |
Human hepatocellular carcinoma HepG2 cells (ATCC HB-6065) were used in all the experiments. HepG2 cells were maintained as a monolayer culture in 95% humidity, atmosphere with 5% of CO2 and 37 ºC. HepG2 cells were passaged at preconfluent densities by the use of trypsin-EDTA solution. Cells were cultured and passaged in Minimal Essential Medium (MEM) supplemented with 10% of Foetal Bovine serum, 1% penicillin/streptomycin, 1% sodium-pryruvate and 1% non-essential amino acids. All media compounds were obtained from Gibco BRL (Breda, the Netherlans). Three milliliters of cells (1x105/mL) were seeded into each well in a 6-wells microtiter plate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated after 6, 12, 24 and 48 hours of incubation with BaP or DMSO in HepG2 total RNA was isolated from cells using miRNeasy mini Kit (Qiagen Westburg bv, Leusden, the Netherlands) according to the manufacturer’s instructions and followed by a DNAse I (Qiagen Inc.) treatment. RNA quantity was measured on a spectrophotometer and quality was determined on a BioAnalyzer (Agilent Technologies, Breda, the Netherlands). Only RNA samples which showed clear 18S and 28S peaks and with a RIN level higher than 8 were used.
|
| Label |
Hy3
|
| Label protocol |
cDNA was generated using 1 μg of total RNA per sample. RNA was labeled only with Hy3 containing dye using the microRNACURY LNA™ microRNA Hy3 Power labeling kit (Exiqon, Denmark) according to the manufacturer’s protocol in a total volume of 25 μl. To adjust the volume to 100 μl, a hybridization buffer provided in miRCURY LNA™ microRNA Array, 5th generation kit (Exiqon, Denmark) was used.
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| |
|
| Hybridization protocol |
In a total volume of 100 μl the sample was incubated for 5 minutes at 95oC, spinned for 2 minutes at maximal speed and injected in the hybridization station. Hybridization was performed in a Tecan HS4800 Pro Hybridization station according to manufacturer protocol.
|
| Scan protocol |
After hybridization the microarray slides were washed accordingly Exiqon instructions. Then arrays were scanned using the GenePix 4000A scanner (Axon Instruments, Foster City, CA). To quantify the signals, the images were processed to TXT files through the GenePix Pro Sofware Suite version 3.0 by using the GAL file for probe annotation as obtained from Exiqon. The Gal file is based on miRBase version 15.0. These TXT files were then imported in R 2.11.0, for quality control and statistical analysis. R package is freely available for academic use from the Comprehensive R Archive Network (http://www.cran.r-project.org/).
|
| Description |
bap-1-48h-Ex2 _12X_ 14180253.gpr
cDNA was generated using 1 μg of total RNA per sample and hybridize in the miRCURY (locked-nuclei acid array) 5th generation arrays (Exiqon, Denmark) that contain 9360 reporters, each present four times on the microarray. In our analysis 1326 human and viral reporters were used, of which 880 are unique human mature microRNAs.
|
| Data processing |
First, the quality of all arrays was inspected using arrayQC, an in-house quality control pipeline that generates virtual images, boxplots, correlation plots, clustering images, MvA and PCA plots. Spike-ins were used for all arrays as an extra quality check. Within this dataset, no arrays were technically deviating. Quantile normalization was applied.
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| |
|
| Submission date |
Aug 08, 2011 |
| Last update date |
Aug 07, 2012 |
| Contact name |
Daneida Lizarraga |
| E-mail(s) |
[email protected]
|
| Fax |
+31 43 3884146
|
| URL |
http://www.toxicogenomics-um.nl/Contact
|
| Organization name |
Maastricht University
|
| Department |
Toxicogenomics
|
| Street address |
Universiteitssingel 50
|
| City |
Maastricht |
| State/province |
Limburg |
| ZIP/Postal code |
6229ER |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL11432 |
| Series (1) |
| GSE31252 |
Benzo[a]pyrene-induced changes in microRNA-mRNA networks |
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