|
| Status |
Public on Jan 14, 2011 |
| Title |
OAW42 siRNA ADRM1(B) vs OAW42 no added RNA (A) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
OAW42 treated with siRNA to ADRM1 (B)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: OAW42
protocol: ADRM1 knockdown
|
| Growth protocol |
Cells were cultured in DMEM medium plus 10% FBS and 4.5 g/L glucose. Treatment: Used mix of four interfering RNAs specific for ADRM1 (from Dharmacon) as a Smartpool in addition to a non-targeting siRNA oligo to use as a control. Transfection was performed using 0.4% Dharmafect transfection reagent and 10nM siRNA according to the manufacturer. OAW42 cells were seeded in 6-well plates and transfected at 60% confluency in antibiotic-free DMEM medium supplemented with 1% L-glutamine, 0.91 ug/ml bovine insulin, 4.5g/1L glucose, and 3.7 g/L sodium bicarbonate). Treated for 48 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen Rneasy Kit following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
|
| |
|
| Channel 2 |
| Source name |
OAW42 untreated
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: OAW42
protocol: untreated (control)
|
| Growth protocol |
Cells were cultured in DMEM medium plus 10% FBS and 4.5 g/L glucose. Treatment: Used mix of four interfering RNAs specific for ADRM1 (from Dharmacon) as a Smartpool in addition to a non-targeting siRNA oligo to use as a control. Transfection was performed using 0.4% Dharmafect transfection reagent and 10nM siRNA according to the manufacturer. OAW42 cells were seeded in 6-well plates and transfected at 60% confluency in antibiotic-free DMEM medium supplemented with 1% L-glutamine, 0.91 ug/ml bovine insulin, 4.5g/1L glucose, and 3.7 g/L sodium bicarbonate). Treated for 48 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen Rneasy Kit following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential
|
| Scan protocol |
Scanned on an Agilent G2505C scanner.
|
| Data processing |
Agilent Feature Extraction Software (v 9.5.1) was used for background subtraction and LOWESS normalization;: (1) local background used, (2) special detrend OFF, (3) adjust background globally OFF, (4) red autoestimate OFF, and (5) green autoestimate OFF.
Rosetta Resolver (v.7.2) was used for data analysis
|
| |
|
| Submission date |
Jan 14, 2011 |
| Last update date |
Jan 14, 2011 |
| Contact name |
Charles L Ginther |
| E-mail(s) |
ginther@ucla.edu
|
| Phone |
310-586-2667
|
| Organization name |
UCLA
|
| Department |
Hem-Onc
|
| Lab |
Slamon
|
| Street address |
2825 Santa Monica Blvd, #200
|
| City |
Santa Monica |
| State/province |
CA |
| ZIP/Postal code |
90404 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE26634 |
Expression changes following siRNA knockdown of ADRM1 in ovarian cell line OAW42 |
|
