|
| Status |
Public on Feb 22, 2012 |
| Title |
CPTR treated CEM-ss |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
CPTR treated CEM-ss
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Human T4-Lymphoblastoid cell line (NIH, Cat. No. 776)
treatment: treated with CPTR
|
| Treatment protocol |
2x10^6 CEM-ss cells were starved for two hours in serum-free medium and subsequently treated with or without 1 µM cell permeable Tre-recombinase (CPTR) for 5 h. After protein transduction cells were maintained for 48 h in normal growth medium prior to RNA extraction.
|
| Growth protocol |
Cells were maintained in RPMI 1640 ( 89%; PSN antibiotics (Gibco), 1%; fetal bovine serum, 10%).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Total RNA was labeled as instructed by the Agilent Two-color Microarray-Based Gene Expression Analysis protocol (Version 5.0.1, August 2006). Labeling of 1 µg control and sample RNA was performed using the Agilent two-color Quick Amp Labeling Kit (5190-0444).
|
| |
|
| Channel 2 |
| Source name |
Untreated CEM-ss
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Human T4-Lymphoblastoid cell line (NIH, Cat. No. 776)
treatment: untreated
|
| Treatment protocol |
2x10^6 CEM-ss cells were starved for two hours in serum-free medium and subsequently treated with or without 1 µM cell permeable Tre-recombinase (CPTR) for 5 h. After protein transduction cells were maintained for 48 h in normal growth medium prior to RNA extraction.
|
| Growth protocol |
Cells were maintained in RPMI 1640 ( 89%; PSN antibiotics (Gibco), 1%; fetal bovine serum, 10%).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Total RNA was labeled as instructed by the Agilent Two-color Microarray-Based Gene Expression Analysis protocol (Version 5.0.1, August 2006). Labeling of 1 µg control and sample RNA was performed using the Agilent two-color Quick Amp Labeling Kit (5190-0444).
|
| |
|
| |
| Hybridization protocol |
Equal amounts of control and sample RNA (825 ng each) were competitively hybridized. The array was hybridized for 17 h at 65°C rotating at 10 rpm. Arrays were washed as instructed by the Agilent Two-color Microarray-Based Gene Expression Analysis protocol (Version 5.0.1, August 2006).
|
| Scan protocol |
Arrays were scanned on a GenePix Personal 4100A scanner (Axon Instruments).
|
| Description |
treated with CPTR vs. untreated CEM-ss cells
|
| Data processing |
Analysis and normalization was conducted using the GenePix Pro 6.0 software (Axon Instruments). Briefly, the DNA gene expression array grid template was adjusted to the array and subsequently all features were aligned and analyzed. Integrated settings were further used to flag features as 'good' when fulfilling the 'fair feature' criteria. For normalization all 'good' flagged spots were included, whereas control spots were excluded [11,111 ID_REFs]. Normalization was performed so that the mean of the ratio of medians of all normalization features is equal to 1. The standard deviation of all spots was calculated using Excel. A twofold change in the expression level was selected as cut off to determine differentially expressed genes.
|
| |
|
| Submission date |
Oct 05, 2011 |
| Last update date |
Feb 22, 2012 |
| Contact name |
Nicole Walz |
| Organization name |
Heinrich-Pette-Institut
|
| Street address |
Martinistr. 52
|
| City |
Hamburg |
| ZIP/Postal code |
20251 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE32643 |
Influence of cell permeable Tre-recombinase (CPTR) treatment on T-cell gene expression |
|
