|
| Status |
Public on Nov 30, 2006 |
| Title |
Mc DKK1 2hrs - mAdbID:60962 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
control
|
| Organism |
Homo sapiens |
| Characteristics |
Tissue: skin
Cell type: Normal human melanocytes
|
| Growth protocol |
Sample Growth Protocol
Other: Neonatal human foreskin melanocytes were obtained from Cascade Biologics, Inc. (Portland, OR). Melanoctye cultures were grown in melanocyte growth medium (MGM) consisting of Medium 154 (Cascade Biologics, Inc.) and HMGS (Cascade Biologics, Inc.). Melanocytes from the third and fifth passages were used in the experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sample Extraction Protocol
Other: Used an RNeasy mini kit (Qiagen, Valencia, CA)
|
| Label |
cy5
|
| Label protocol |
Cy5 Sample Labeling Protocol - generic
|
| |
|
| Channel 2 |
| Source name |
DKK1
|
| Organism |
Homo sapiens |
| Characteristics |
Tissue: skin
Cell type: Normal human melanocytes
|
| Treatment protocol |
Treatment type: compound
Agent: recombinant human dickkopf 1 (rhDKK1)
Treatment dose: 50 ng/ml
Treatment time: 5 days
Treatment temperature: 37 C
|
| Growth protocol |
Sample Growth Protocol
Other: Neonatal human foreskin melanocytes were obtained from Cascade Biologics, Inc. (Portland, OR). Melanoctye cultures were grown in melanocyte growth medium (MGM) consisting of Medium 154 (Cascade Biologics, Inc.) and HMGS (Cascade Biologics, Inc.). Melanocytes from the third and fifth passages were used in the experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sample Extraction Protocol
Other: Used an RNeasy mini kit (Qiagen, Valencia, CA)
|
| Label |
cy3
|
| Label protocol |
Cy3 Sample Labeling Protocol - generic
|
| |
|
| |
| 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 42øC 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 ¶l COT-1 DNA (8-10 ug/ul) and 1 ul poly A (8-10 ug/ul). Denature target at 100øC 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 42øC 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 42øC 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")
|
| Description |
mAdb experiment ID: 60962
|
| Data processing |
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 |
Aug 14, 2006 |
| Last update date |
Jul 19, 2010 |
| Contact name |
Vincent Hearing |
| E-mail(s) |
hearingv@nih.gov
|
| Organization name |
National Institutes of Health
|
| Department |
National Cancer Institute
|
| Lab |
Laboratory of Cell Biology
|
| Street address |
Bldg 37 Rm 2132
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL1528 |
| Series (1) |
| GSE5515 |
Effects of DKK1 on gene expression on melanocytes |
|
| Data table header descriptions |
| ID_REF |
NCI mAdb well id plus replicate number |
| VALUE |
log ratio (log2 of PRE_VALUE) |
| 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 |
