|
| Status |
Public on Jun 01, 2009 |
| Title |
OCI-Ly3 Dox 3 days + Ikk Inhibitor 24 hours - mAdbID:81135 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
OCI-Ly3 - Ikk Inhibitor (24 hours)
|
| Organism |
Homo sapiens |
| Characteristics |
Cell line: OCI-Ly3 Cell Line
|
| Treatment protocol |
Treatment type: compound
Agent: Ikk Inhibitor (MLN120b)
Treatment dose: 12.5 uM (Ikk Inhibitor)
Treatment time: 24 hr (Ikk Inhibitor)
In-vivo treatment: Treated 24 hours of Ikk inhibitor (MLN120b).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Reagent Extraction Protocol
Extraction method: Trizol Reagent
Other: Total RNA was prepared using the Trizol reagent (Gibco BRL) according to manufacturer's instructions.
|
| Label |
cy3
|
| Label protocol |
Cy3 Labeling Protocol
Other: Isolated RNA (50 ug) was reverse transcribed by oligo dT-primed polymerization using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). The targets were coupled to Cy3-conjugated deoxynucleotides (Amersham Biosciences, Piscataway, NJ) for 1 hour at room temperature. Probes were combined and concentrated using Microcon YM-30 columns (Millipore, Billerica, MA).
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| |
|
| Channel 2 |
| Source name |
OCI-Ly3 - Dox 3 days + Ikk Inhib 24 hrs
|
| Organism |
Homo sapiens |
| Characteristics |
Cell line: OCI-Ly3 Cell Line
|
| Treatment protocol |
Treatment type: compound
Agent: Doxycycline; Ikk Inhibitor (MLN120b)
Treatment dose: 20 ng/ml (Dox); 12.5 uM (Ikk Inhibitor)
Treatment time: 3 days (Dox); 24 hr (Ikk Inhibitor)
In-vivo treatment: Treated 2 days with Doxycycline followed by 24 hours of Ikk inhibitor (MLN120b).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Reagent Extraction Protocol
Extraction method: Trizol Reagent
Other: Total RNA was prepared using the Trizol reagent (Gibco BRL) according to manufacturer's instructions.
|
| Label |
cy5
|
| Label protocol |
Cy5 Labeling Protocol
Other: Isolated RNA (50 ug) was reverse transcribed by oligo dT-primed polymerization using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). The targets were coupled to Cy5-conjugated deoxynucleotides (Amersham Biosciences, Piscataway, NJ) for 1 hour at room temperature. Probes were combined and concentrated using Microcon YM-30 columns (Millipore, Billerica, MA).
|
| |
|
| |
| Hybridization protocol |
NCI cDNA Microarray Hybridization
Other: For pre-hybrization, apply 40 ul of pre-hybridization buffer (5X SSC, 0.1% SDS, 1% BSA) to the array and incubate at 42°C for at least 30 minutes and up to an hour. Wash off the pre-hybridization solution by rapidly plunging the slide in distilled water for 2 minutes, then transfer slide to 100% isopropanol for 2 minutes. Allow slide to air dry completely prior to use. (Can spin dry if in a rush.) (NOTE: Do not exceed 1 hour after pre-hybridization/drying before setting up hybridization.) For hybridization, combine Cy3 and Cy5 labeled targets together (~9 ul recovered for each). Add 1 µl COT-1 DNA (8-10 ug/ul) and 1 ul poly A (8-10 ug/ul). Denature target at 100°C for 1 minute, then snap cool on ice. (Final volume should be about 20 ul.) Make fresh 2X Formamide hybridization buffer (50% formamide, 10X SSC, 0.2% SDS) and warm to 42°C just before adding to samples. Add 20 ul of 2X F-hyb buffer to samples. Load 40 ul sample onto microarray. Add 20 ul of 3X SSC to wells in hyb chamber to maintain humidity. Incubate overnight (12-16 hours) at 42°C in water bath or hybridization oven. After hybridization of slides, wash slides for 2 minute in 2X SSC with 0.1% SDS (with occasional plunging), for 2 minute in 1X SSC (with occasional plunging), for 2 minutes in 0.2X SSC (with occasional plunging), for 1 minute in 0.05X SSC, and spin for 3 minutes at 650 rpm to dry. (Refer to "NCI Microarray Manual")
|
| Scan protocol |
Creator: GenePix Pro 3.0.6.89
Scanner: GenePix 4000A [55478]
PMTVolts: 850;; 750
ScanPower: 100;; 100
LaserPower: 1.7655;; 2.24464
Temperature: 37.3582
|
| Description |
mAdb experiment ID: 81135
|
| Data processing |
mAdb Data Processing Protocol (v. 1)
Calculation Method: After background correction and removal of flagged values, log base 2 expression ratios were median centered and linear transformed to obtain the log and linear values given in the data table.
|
| |
|
| Submission date |
Jun 26, 2008 |
| Last update date |
Jun 26, 2008 |
| Contact name |
Louis M. Staudt |
| E-mail(s) |
lstaudt@mail.nih.gov
|
| Phone |
301-402-1892
|
| Organization name |
National Cancer Institute
|
| Department |
Lymphoid Malignancies Branch
|
| Lab |
Louis M Staudt
|
| Street address |
9000 Rockville Pike, Bldg 10, Rm 4N114
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL3278 |
| Series (1) |
| GSE11902 |
Compensatory IKKa activation of classical NF-kB signaling during IKKb inhibition |
|
| Data table header descriptions |
| ID_REF |
NCI mAdb well id plus replicate number |
| VALUE |
log ratio (log2 of PRE_VALUE) but with flagged values removed |
| PRE_VALUE |
Calibrated Ratio (CY5 channel/CY3 channel) |
| Slide_block |
Array block location |
| Slide_column |
Array column location |
| Slide_row |
Array row location |
| CY5_mean |
Red Channel Sample mean Signal (Background Subtracted) |
| CY5_SD |
Red Channel Sample Standard Deviation |
| CY5_BKD_median |
Red Channel Sample median Background Level |
| CY5_BKD_SD |
Red Channel Sample Background Standard Deviation |
| CY3_mean |
Green Channel Sample mean Signal (Background Subtracted) |
| CY3_SD |
Green Channel Sample Standard Deviation |
| CY3_BKD_median |
Green Channel Sample median Background Level |
| CY3_BKD_SD |
Green Channel Sample Background Standard Deviation |
| Flag |
Quality flag 0->good, -50->Not found, -100->Bad |
| UNF_VALUE |
log ratio (log2 of PRE_VALUE) |
