|
| Status |
Public on Jun 25, 2015 |
| Title |
Nanopillar-cultured hepatocytes 2 |
| Sample type |
RNA |
| |
|
| Source name |
Nanopillar-cultured hepatocytes extracted from rat #2
|
| Organism |
Rattus norvegicus |
| Characteristics |
treatment: Nanopillar-cultured
cell type: primary hepatocyte
genetic background: F344/DuCrlCrlj
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the four kinds of samples mentioned above using an RNeasy Plus Micro Kit (Qiagen GmbH, Germany) according to the manufacturer’s instructions. Quantity and quality of RNA were determined by a Nanodrop ND-1000 spectrophometer (Thermo Fisher Scientific Inc., Waltham, MA) and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA), as recommended.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was amplified and labeled with Cyanine 3 (Cy3) using an Agilent Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies, Palo Alto, CA) following the manufacturer's instructions. Briefly, 100 ng of total RNA was reverse-transcribed to double-strand cDNA by using a poly dT-T7 promoter primer. Primer, template RNA, and quality-control transcripts of known concentration and quality were first denatured at 65ºC for 10 min and incubated for two hours at 40ºC with 5X first-strand buffer, 0.1-M DTT, 10-mM dNTP mix, and AffinityScript RNase Block Mix. The AffinityScript enzyme was inactivated at 70ºC for 15 min. cDNA products were then used as templates for in vitro transcription to generate fluorescent cRNA. cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3 labeled-CTP and incubated at 40ºC for two hours. The labeled cRNAs were purified using Qiagen’s RNeasy mini spin columns and eluted in 30 μl of nuclease-free water. Subsequently, amplified cRNA quantity and labeled cyanine incorporation were determined using a Nanodrop ND-1000 spectrophometer and an Agilent bioanalyzer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labeled cRNA was fragmented and hybridized at 65ºto the Agilent Whole Rat Genome Microarrays 4× 014879).
|
| Scan protocol |
Microarrays were scanned using an Agilent DNA microarray scanner.
|
| Description |
Gene expression in nanopillar-cultured hepatocytes
|
| Data processing |
The microarray data were analyzed by using the GeneSpring GX 10.0.2. (Agilent Technologies)
Intensity values of each scanned feature were quantified using Agilent Feature Extraction Software version 10.5.1.1, which performs background subtractions.
|
| |
|
| Submission date |
Jun 26, 2012 |
| Last update date |
Jun 25, 2015 |
| Contact name |
Ryosuke Takahashi |
| E-mail(s) |
ryosuke.takahashi.jr@hitachi.com
|
| Organization name |
Hitachi, Ltd.
|
| Department |
Central Research Laboratory
|
| Lab |
Advanced Research Department
|
| Street address |
2520 Hatoyama
|
| City |
Saitama |
| ZIP/Postal code |
3500395 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7294 |
| Series (1) |
| GSE38950 |
Comprehensive expression analysis of primary hepatocytes under four different culture conditions |
|
