|
| Status |
Public on Sep 01, 2007 |
| Title |
Smad2-independent genes in BxPC-3 cells |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
S2KD (Smad2 knockdown) cells with TGFb stimulation
|
| Organism |
Homo sapiens |
| Characteristics |
stable Smad2 knockdown BxPC-3 cells
|
| Treatment protocol |
The cells were stimulated by incubation in the presence (+) of 10 ng/ml of TGF-β for 4 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA).
|
| Label |
Cy5/Cy3
|
| Label protocol |
Fluorescent dye (Cy5 and Cy3-dCTP, Amersham Pharmacia Biotech, Piscataway, NJ) labeled DNA was produced through Eberwine's linear RNA amplification method.and subsequent enzymatic reaction. the amplified RNA (aRNA) was purified with RNeasy Mini Kit (Qiagen). Because sense oligonucleotide arrays were used here, we took a cDNA labeling approach with Klenow enzyme after reverse transcription.
|
| |
|
| Channel 2 |
| Source name |
S2KD (Smad2 knockdown) cells without TGFb stimulation
|
| Organism |
Homo sapiens |
| Characteristics |
stable Smad2 knockdown BxPC-3 cells
|
| Treatment protocol |
The cells were stimulated by incubation in the absence (-) of 10 ng/ml of TGF-β for 4 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA).
|
| Label |
Cy3/Cy5
|
| Label protocol |
Fluorescent dye (Cy3 and Cy5-dCTP, Amersham Pharmacia Biotech, Piscataway, NJ) labeled DNA was produced through Eberwine's linear RNA amplification method.and subsequent enzymatic reaction. the amplified RNA (aRNA) was purified with RNeasy Mini Kit (Qiagen). Because sense oligonucleotide arrays were used here, we took a cDNA labeling approach with Klenow enzyme after reverse transcription.
|
| |
|
| |
| Hybridization protocol |
Labeled control and test samples were quantitatively adjusted based on the efficiency of Cy-dye incorporation and mixed into 80 µL hybridization solution (3×SSC, 0.2% SDS, 25% formamide and 5×Denhart’s). DNA in hybridization solution was denatured at 95℃ for 3 min prior loading on a microarray. Hybridization was performed under LifterSlipTM (Erie Company), which allows for even dispersal of hybridization solutions between the microarray and coverslip. The hybridization chamber was placed on a 3D-Tilting Agitator (CapitalBio Corp.) to facilitate the microfluidic circulation under the coverslip. The array was hybridized at 42℃ overnight and washed with two consecutive washing solutions (0.2% SDS, 2×SSC at 42℃ for 5 min, and 0.2% SSC for 5 min at room temperature).
|
| Scan protocol |
Microarrays were scanned with a confocal LuxScanTM scanner (CapitalBio Corp.), and the data of obtained images were extracted with SpotData Software (CapitalBio Corp.).
|
| Description |
Confirmation of the Smad2-independent genes via TGFb stimulation in BxPC-3 cells.
|
| Data processing |
The raw data was normalized using a space and intensity-dependant LOWESS program (Yang et al., 2002). The data from faint spots was removed, in which the intensity was lower than the average intensity plus 2 standard deviations of the negative controls on the array.
|
| |
|
| Submission date |
Jul 13, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
yu jian |
| E-mail(s) |
yujian@capitalbio.com
|
| Phone |
(86)-10-80726868
|
| Fax |
(86)-10-62773059
|
| Organization name |
Tsinghua University
|
| Department |
Medical Systems Biology Research Center
|
| Street address |
Qinghuayuan
|
| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100084 |
| Country |
China |
| |
|
| Platform ID |
GPL4525 |
| Series (1) |
| GSE8464 |
Invasion control by a positive feedback loop mechanism Smad4-null cells |
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