|
| Status |
Public on Jan 30, 2009 |
| Title |
NHMEC 99005 - dye swap - replicate 3 - mAdbID:53691 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NHMEC 99005 (untreated) - Cy5
|
| Organism |
Homo sapiens |
| Characteristics |
Gender: female
Tissue: mammary gland
Cell line: NHMEC 99005
Cell type: Normal Human Mammary Epithelial
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Extraction Protocol
Other: For isolation of RNA the cells were lysed with 1.0 mL of Trizol Reagent (Invitrogen Corp., Carlsbad, CA) and RNA was extracted according to manufacturer’s protocol.
|
| Label |
cy5
|
| Label protocol |
Cy5 Labeling Protocol
Other: Labeled with Cy5 by indirect coupling.
|
| |
|
| Channel 2 |
| Source name |
NHMEC 99005 - TAM - Cy3
|
| Organism |
Homo sapiens |
| Characteristics |
Gender: female
Tissue: mammary gland
Cell line: NHMEC 99005
Cell type: Normal Human Mammary Epithelial
|
| Treatment protocol |
Treatment type: compound
Agent: Tamoxifen
Treatment dose: 10 uM
Treatment time: 48 hours
Treatment temperature: 37C
Other: Cells at passage 13 were plated at 1 x10^6 cells/15 cm plate or T-175 flask for DNA preparation, and at 1x10^6 cells/6-well plate for RNA preparation and survival, and grown for 48 hours prior to treatment with 10 uM TAM in vehicle (DMSO) for an additional 48 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Extraction Protocol
Other: For isolation of RNA the cells were lysed with 1.0 mL of Trizol Reagent (Invitrogen Corp., Carlsbad, CA) and RNA was extracted according to manufacturer’s protocol.
|
| Label |
cy3
|
| Label protocol |
Cy3 Labeling Protocol
Other: Labeled with Cy3 by indirect coupling.
|
| |
|
| |
| Hybridization protocol |
NCI Oligo 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 ul 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), and spin for 3 minutes at 650 rpm to dry. (Refer to "NCI Microarray Manual")
|
| Scan protocol |
Creator: GenePix Pro 4.0.0.54
Scanner: GenePix 4000B [84573]
ScanPower: 100;; 100
LaserPower: 3.37321;; 3.85022
Temperature: 24.5262
|
| Description |
mAdb experiment ID: 53691
|
| 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 |
Nov 05, 2008 |
| Last update date |
Jan 30, 2009 |
| Contact name |
Ofelia Ana Olivero |
| E-mail(s) |
oliveroo@exchange.nih.gov
|
| Phone |
3014357843
|
| Organization name |
NCI-CCR-
|
| Department |
CDI
|
| Lab |
LCBG
|
| Street address |
37 Convent Dr-MSC 4255
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL1528 |
| Series (1) |
| GSE13476 |
Tamoxifen induces expression of immune-response related genes in cultured NHMEC |
|
| Data table header descriptions |
| ID_REF |
NCI mAdb well id plus replicate number |
| VALUE |
log ratio(log2 of Cy3/Cy5 channel) 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) |
| INV_VALUE |
same as UNF_VALUE but with flagged values removed |
