|
| Status |
Public on Feb 13, 2010 |
| Title |
PC12 control rep4 |
| Sample type |
RNA |
| |
|
| Source name |
PC12, Continuous complete medium stimulation, 3 hr, replicate 4
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: PC12
|
| Treatment protocol |
For stimulants treatment, PC12 cells were plated on poly-l-lysine-coated dishes in complete medium for 24 hours and then switched to complete medium supplemented with the 50 ng/ml of NGF (R&D), 100 nM of PACAP (Sigma), 50 ng/ml of bFGF (Sigma), 50 ng/ml of EGF (Roche), 10 nM of insulin (Sigma) or 10 μM of forskolin (Sigma). For pharmacological inhibition of MEK1 and PI3K activity, cultures were pretreated with 50 μM U0126 (Promega) and 50 μM LY294002 (Sigma) respectively, for 20 minutes before addition of NGF. For washing of extension factors or inhibitors in compounds containing culture medium, the culture medium was replaced with an equal volume of complete medium four times.
|
| Growth protocol |
PC12 cells were maintained in Dulbecco’s modified Eagle’s medium containing 5% horse serum and 10% bovine calf serum （Nichirei） at 5% CO2, 37℃.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified with the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. The concentration of isolated RNA was determined by an ND-1000 spectrophotometer (Nanodrop), according to manual instructions. Integrity and quantity of isolated total RNA were assessed by the Agilent 2100 Bioanalyzer (Agilent), according to manual instructions.
|
| Label |
Cy3
|
| Label protocol |
Six hundred nanograms of total RNA was converted into labeled complementary RNA (cRNA) with nucleotides coupled to a fluorescent dye (Cy-3) by means of the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). The quality and quantiy of the resulting labeled cRNA were assessed with an ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60℃ for 30 minutes in a reaction volume of 55 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Rat Genome Oligo Microarrays (G4131F) for 17 hours at 65℃ in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37℃ GE Wash buffer 2 (Agilent), then dried.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of 3 hours after complete medium stimulation
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 014879_D_20070207) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Sep 08, 2009 |
| Last update date |
Feb 12, 2010 |
| Contact name |
Jaehoon Chung |
| E-mail(s) |
souhoon@gmail.com
|
| Organization name |
Tokyo university
|
| Department |
Department of Biophysics and Biochemistry
|
| Lab |
Kuroda Lab
|
| Street address |
7-3-1 Hongo
|
| City |
Bunkyo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
113-0033 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4135 |
| Series (1) |
| GSE18016 |
Identification of genes which are responsible for induction of the initial differentiation process of PC12 cells |
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