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| Status |
Public on Mar 27, 2017 |
| Title |
Tmub1-knockdown |
| Sample type |
RNA |
| |
|
| Source name |
Cell line, Lentivirus of Tmub1-knockdown transfected, replicate 1
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: BRL-3A
|
| Treatment protocol |
BRL-3A cells were divided into five groups and cultured as follows: Tmub1 overexpression lentivirus-transduced (Lv-Tmub1(+)), Tmub1 knockdown lentivirus-transduced (Lv-Tmub1(-)), Lv-Tmub1(+)-Negative Control, Lv-Tmub1(-)-Negative Control and normal control. The cells were harvested 48 h post-transduction and total RNA for microarray analysis was extracted using an extraction reagent (TRIzol; Invitrogen)
|
| Growth protocol |
Normal rat hepatocyte cells (BRL-3A, Cell Bank of Chinese Academy of Sciences, Shanghai, China) were maintained in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen Life Technologies), 100 units/ml penicillin, and 100 µg/ml streptomycin, and cultured in a humidified atmosphere of 5% CO2 at 37 °C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were harvested 48 h post-transduction and total RNA for microarray analysis was extracted using an extraction reagent (TRIzol; Invitrogen). Then, complementary DNA was synthesized and labeled before it was purified and hybridized to the microarray (Arraystar, Rockville, MD).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1 ug RNA using the Quick Amp Labeling Kit, One-Color (Agilent p/n 5190-0442) according to the manufacturer's instructions, followed by RNeasy Mini Kit (Qiagen p/n 74104). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.5 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 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Microarray (Agilent-014879) Hybridization Chambers (Agilent p/n G2530-60029) 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 immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Microarray Scanner (Agilent p/n G2565BA) 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 after Lentivirus of Tmub1-knockdown transfected
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Mar 26, 2017 |
| Last update date |
Mar 27, 2017 |
| Contact name |
Xu jianhua |
| E-mail(s) |
[email protected]
|
| Organization name |
TMMU
|
| Street address |
Daping
|
| City |
Chongqing |
| ZIP/Postal code |
400012 |
| Country |
China |
| |
|
| Platform ID |
GPL14746 |
| Series (1) |
| GSE97040 |
The role of Tmub1 protein in the regulation of hepatic cell transcription |
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