|
| Status |
Public on May 22, 2012 |
| Title |
IRF9-RNAi and IFN-alpha2c OVCAR3 - repeat 3 - mAdbID:90205 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Negative RNAi OVCAR3
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: ovary
cell line: OVCAR3
cell type: human ovarian adenocarcinoma cells
disease state: human ovarian adenocarcinoma
|
| Treatment protocol |
Treatment type: small molecule
Agent: Stealth RNAi Negative Control Low GC (Invitrogen)
Treatment dose: 100 nM
Treatment time: 4 hours
Treatment temperature: 37C
In-vitro treatment: Stealth RNAi Negative Control Low GC (Invitrogen) was used as negative-RNAi. The RNAi oligos (100nM) were mixed with Lipofectamine (Invitrogen) in OPTI-MEM medium (Invitrogen), and the mixture was added to the medium on cells. The cells were incubated for 4 h at 37°C then the medium was replaced with medium containing antibiotics.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sample Extraction Protocol
Other: Total RNA was purified using RNeasy Kit (Qiagen; Valencia, CA).
|
| Label |
cy3
|
| Label protocol |
Cy3 Labeling Protocol
Other: Amino allyl-oligo-dT (5’ amino-modified) primer (1µg/µl) (31) was incubated with 5 µg total RNA and then reverse transcription (RT) labeling reaction mixture from FairPlay Microarray Labeling Kit (Agilent Technologies) was added to each sample. Following incubation at 42°C for 90 min, cDNA was purified using MinElute PCR Purification columns (Qiagen). After eluting and drying cDNA samples, they were resuspended in 5µl 2× Coupling Buffer. Following, 5µl of Cy3 mono-reactive dye (GE Healthcare/Amersham Biosciences; Piscataway, NJ) was added to the samples and incubated in the dark at room temperature for 90 min.
|
| |
|
| Channel 2 |
| Source name |
IRF9-RNAi and IFN-aplha2c OVCAR3
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: ovary
cell line: OVCAR3
cell type: human ovarian adenocarcinoma cells
disease state: human ovarian adenocarcinoma
|
| Treatment protocol |
Treatment type: small molecule
Agent: human interferon (IFN-apha2c)
Treatment dose: 10 ng/ml
Treatment time: 24 hours
Treatment temperature: 37C
In-vitro treatment: OVCAR3 cells were plated and adhered overnight at 37°C. 10 ng/ml of IFN-apha2c was then added into the medium. After incubation for 24 hours at 37°C, the cells were harvested and used for microarray analysis.
Treatment type: small molecule
Agent: IFN regulatory factor (IRF) 9 RNA interference (IRF9-RNAi)
Treatment dose: 100 nM
Treatment time: 4 hours
Treatment temperature: 37C
In-vitro treatment: Duplex RNAi, 25 base pair (bp) and 21 bp, were purchased from Invitrogen and Dharmacon (Lafayette, CO), respectively. The RNAi oligos (100 nM) were mixed with Lipofectamine (Invitrogen) in OPTI-MEM medium (Invitrogen), and the mixture was added to the medium on cells. The cells were incubated for 4 h at 37°C then the medium was replaced with medium containing antibiotics.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sample Extraction Protocol
Other: Total RNA was purified using RNeasy Kit (Qiagen; Valencia, CA).
|
| Label |
cy5
|
| Label protocol |
Cy5 Labeling Protocol
Other: Amino allyl-oligo-dT (5’ amino-modified) primer (1µg/µl) (31) was incubated with 5 µg total RNA and then reverse transcription (RT) labeling reaction mixture from FairPlay Microarray Labeling Kit (Agilent Technologies) was added to each sample. Following incubation at 42°C for 90 min, cDNA was purified using MinElute PCR Purification columns (Qiagen). After eluting and drying cDNA samples, they were resuspended in 5µl 2× Coupling Buffer. Following, 5µl of Cy5 mono-reactive dye (GE Healthcare/Amersham Biosciences; Piscataway, NJ) was added to the samples and incubated in the dark at room temperature for 90 min.
|
| |
|
| |
| Hybridization protocol |
Hybridization Protocol
Other: Using MinElute PCR Purification columns, dye coupled cDNA samples were purified and then respective samples were mixed together, heated at 95°C for 5 min followed by incubation at 42°C for 5 min. SlideHyb Buffer #1 (Ambion; Austin, TX) was added to the combined samples, and then samples were loaded onto ~21K 70mer oligonucleotide microarrays (printed by Genomics Technology Section RTB/NIAID) sealed with MAUI Mixer FL hybridization chambers (BioMicro Systems; Salt Lake City, UT) and incubated overnight at 42°C. Arrays were then washed for 5 min each in buffers; Buffer #1: 1× saline sodium citrate (SSC) with 0.05% SDS; Buffer #2: 0.1× SSC. Arrays were then dried.
|
| Scan protocol |
Creator: GenePix Pro 5.1.0.19
Scanner: GenePix 4200A 01 [99052]
ScanPower: 20;; 15
LaserPower: 3.2428;; 2.84973
Temperature: 24.5625
Scanning Protocol
Other: Arrays were scanned using an Axon GenePix 4200A microarray scanner with GenePix Pro 5.1 software (Molecular Devices; Sunnyvale, CA).
|
| Description |
mAdb experiment ID: 90205
|
| Data processing |
Data Processing Protocol
Calculation Method: Array data was normalized via 50th percentile normalization and spot intensity was calculated by subtracting local median background from mean foreground intensity.
|
| |
|
| Submission date |
Jun 07, 2011 |
| Last update date |
May 22, 2012 |
| Contact name |
Joseph Bekisz |
| E-mail(s) |
[email protected]
|
| Phone |
301-594-9241
|
| Fax |
301-480-8099
|
| Organization name |
NIH
|
| Department |
NIAID
|
| Lab |
CBS
|
| Street address |
50 South Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL1054 |
| Series (1) |
| GSE29790 |
BID is critical for IFN-alpha-induced cell death |
|
