|
| Status |
Public on Jan 12, 2008 |
| Title |
MCF7_AZT_azaC_3hour_sample1 |
| Sample type |
RNA |
| |
|
| Source name |
MCF7 cells
|
| Organism |
Homo sapiens |
| Characteristics |
MCF7 ERAlpha positive Breast Adenocarcinoma cell line
|
| Biomaterial provider |
ATCC
|
| Treatment protocol |
MCF7 cells at 80% confluency were treated with 5 micromolar azacytidine for two hours
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed in 10 ml Trizol, incubated for 5 min at room temperature. After addition of 1 ml chloroform, the lysate was mixed and incubated for 5 min at room temperature. The pahses were seperated by 12000xg for 15 min, the aquous phase was removed and 2-propanol was added. After a 10 min incubation at RT, the tubes were spun at 10000 rpm at 4C for 10 minutes, washed with 75% ethanol and finally, total RNA was dissolved in water and quantified.
|
| Label |
biotin
|
| Label protocol |
Synthesis of cRNA by IVT:
Component 1x reaction
10x T7 reaction buffer 4,0ul
T7 ATP solution 4ul
T7 GTP solution 4ul
T7 CTP solution 4ul
T7 UTP solution 3ul
10mM biotin-16-UTP 7,5ul
10x T7 enzyme mix 4ul
30,5 ul of mixture was added to each sample and incubated at 37C for 14 hours.
Cleanup and Quantification of Biotin Labeled cRNA
Add 60ul Rnase free water and 350 ul buffer RLT to the IVT reaction, mix thoroughly. Add 250 ul ethanol (96-100%), mix well. Apply sample to an RNeasy spin column in a collection tube (2mL tube). Centrifuge for 15 seconds at 8000g (10000rpm). Add 500 ul buffer RPE Wash. Centrifuge for 15 seconds at 8000g (10000rpm). Repeat the last two steps. Replace the 2mL collection tube. Centrifuge for 2 minutes at 8000g (10000rpm). New 1,5mL collection tube: Add 50 ul nuclease-free water onto the filtermembrane. Incubate for 10 minutes at RT. Centrifuge for 1 minute at 8000g (10000rpm). Repeat the last three steps.
|
| |
|
| Hybridization protocol |
Centrifuge samples for 5 sec at max speed. Heat hybridistion cocktail to 90C for 5 min. Cool tubes on ice for 5 min. Load arrays within 30 min of denatured cRNA. Hybridize at 37 C for 18-24 hours. Prepare 0.75x TNT buffer for 46C over night. Wash 3x in TNT covered from light. Put in Cy5-Strep solution, incubate 30 min at RT proteced from light. Repeat TNT washes 4x. Incubate in a large reservoir of 0.1xSSC/0.05%Tween20 for 30 seconds, centrifuge for 3 min at 200 rpm.
|
| Scan protocol |
Expression_5um.gps: whole genome products Expression_10um.gps: 10K and 20K products wavelength: 635 nm PMT voltage: 600 V laser power: 100% pixel size: 5 um for Whole Genome products
|
| Description |
This sample is the 1st sample treated with 5 micromolar azacytidine for three hours
|
| Data processing |
Raw intensity values were median background subtracted and normalized to the median of the chip intensity
|
| |
|
| Submission date |
Jan 10, 2008 |
| Last update date |
Jan 11, 2008 |
| Contact name |
George Reid |
| E-mail(s) |
reid@embl.de
|
| Phone |
0049 6221 387161
|
| Organization name |
European Molecular Biology Organisation
|
| Lab |
Gannon Lab
|
| Street address |
Meyerhofstrasse 1
|
| City |
Heidelberg |
| ZIP/Postal code |
69117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL2895 |
| Series (1) |
| GSE10145 |
Azacytidine time course on MCF7 cells |
|
