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| Status |
Public on Aug 25, 2010 |
| Title |
HT29_2D_day1_replicate1 |
| Sample type |
RNA |
| |
|
| Source name |
HT29_2D_day1_replicate1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: in vitro tumor cells
cell line: HT-29
growth condition: 2D monolayer 1 day
|
| Treatment protocol |
HT-29 cells were seeded in 96-well plates at 1×10^4 cells/100 ul of NanoCulture Medium-M (Scivax) in either NanoCulture plates (NCPs) (Scivax), or in polystyrene plates for 2D monolayers, for 1 day or 7 days. Cells at each time point were collected and used for RNA extraction and DNA microarray analysis.
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| Growth protocol |
HT-29 cells were maintained routinely as 2D monolayers in a humidified atmosphere of 5% CO2 in air at 37 °C in the growth medium recommended by suppliers. Exponentially growing cells were used in experiments, after trypsinization to detach them from the plates. Viable cells were counted using trypan blue dye-exclusion. For experiments, cells were seeded in 96-well plates at 1×10^4 cells/100 ul of NanoCulture Medium-M (Scivax) in either NCPs (Scivax), or in polystyrene plates for 2D monolayers, for 1 day or 7 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNeasy mini kit (QIAGEN, Valencia, CA) following the manufacturer's recommendations. The protocol includes an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ml containing 1x Agilent fragmentation buffer and 10 x 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 Human Genome Oligo Microarrays (G4112F) for 17 hours at 65°C 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°C 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 cells in 2D cluture on day 1
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1-v5_10_Apr08 and Grid: 014850_D_F_20080627) to obtain background subtracted and spatially detrended Processed Signal intensities.
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| |
|
| Submission date |
Aug 24, 2010 |
| Last update date |
Aug 24, 2010 |
| Contact name |
Yukie Yoshii |
| E-mail(s) |
yukiey@u-fukui.ac.jp
|
| Phone |
+81-776-61-8491
|
| Fax |
+81-776-61-8170
|
| Organization name |
University of Fukui
|
| Department |
Biomedical Imaging Research Center
|
| Street address |
23-3, Matsuoka Shimoaizuki
|
| City |
Eiheiji |
| ZIP/Postal code |
910-1193 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE23773 |
Characterization of HT-29 cells cultured in NanoCulture plates |
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