|
| Status |
Public on May 05, 2006 |
| Title |
CCAT DTS Rep A2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Neuroblastoma cell line from ATCC (Cat# CCL-131) transfected with CCAT DTS (280AA C-term of CaV 1.2, Accesion# M67515)
|
| Organism |
Mus musculus |
| Characteristics |
Neuroblastoma cell line from ATCC (Cat# CCL-131) transfected with CCAT (503AA C-term of CaV 1.2, Accesion# M67515) for 24hrs
|
| Treatment protocol |
For the mRNA in the green channel (SIG_1): Neuro2A Cells were transfected using Lipofoctamine 2000 transfection reagent with a PA1 plasmid containing CCAT DTA (280AA C-term of CaV 1.2, Accesion# M67515). 24 hours after transfection, GFP expressing cells were selected using fluorescence activated cell sorting (FACS) and total RNA was extracted from these cells.
|
| Growth protocol |
Cell were maintained acording to the recomendations of ATCC. ATCC complete growth medium: Minimum essential medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10%; Temperature: 37.0C; Atmosphere: air, 95%; carbon dioxide (CO2), 5%
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNAesay Mini kit (Qiagen). RNA sample quality assessment was performed using the Agilent 2100 bioanalyzer and RNA LabChip Kits.
|
| Label |
Cy3
|
| Label protocol |
In an ozone-free room, labeling and linear amplification was accomplished using Agilent’s low RNA Input Linear Amplification Kit PLUS, Two-Color. All steps were performed according to manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
Untransfected Neuro2A Cells
|
| Organism |
Mus musculus |
| Characteristics |
Neuroblastoma cell line from ATCC (Cat# CCL-131)
|
| Treatment protocol |
Untransfected Neuro2A cells were grown under identical conditions to transfected cells and sorted using a fluorescence activated cell sorter to replicate the condition of cells expressing CCAT.
|
| Growth protocol |
Cell were maintained acording to the recomendations of ATCC. ATCC complete growth medium: Minimum essential medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10%; Temperature: 37.0C; Atmosphere: air, 95%; carbon dioxide (CO2), 5%
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNAesay Mini kit (Qiagen). RNA sample quality assessment was performed using the Agilent 2100 bioanalyzer and RNA LabChip Kits.
|
| Label |
Cy5
|
| Label protocol |
In an ozone-free room, labeling and linear amplification was accomplished using Agilent’s low RNA Input Linear Amplification Kit PLUS, Two-Color. All steps were performed according to manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Gene Expression Hybridization Kit, Hybridization chamber-Surehyb and hybridization oven were used for the hybridization step followed by Gene Expression Wash buffers and Stabilization and drying solution (Agilent). All steps were performed according to manufacturer’s instructions. Two color RNA Spike-In kit (Agilent) was used to monitor microarray workflow.
|
| Scan protocol |
Microarrays were scanned using Agilent’s Microarray scanner. Feature extraction was performed using Agilent's feature extraction software 8.5.
|
| Description |
The goal of these experiments was to identify the genes that are regulated by CCAT, a novel transcription factor derived from the C-terminus of CaV1.2. Neuro2A neuroblastoma cells were transfected with the last 503 AA of CaV1.2 which is full length CCAT (CCAT FL) or with the last 280 AA of CaV1.2, a form of CCAT that lacks the transcriptional activation domain (CCAT DTA). The mRNA from either CCAT FL or CCAT DTA expressing cells was hybridized to Agilent mouse genome microarrays along with mRNA from untransfected neuro2A cells. The microarray data was subsequently analyzed with the Rossetta Luminator gene expression data analysis system. Subsequent investigation revealed that CCAT DTA can still activate transcription albeit at much lower levels than CCAT FL.
|
| Data processing |
Microarray results were analyzed using the Rosetta LuminatorTM gene expression data analysis system.
|
| |
|
| Submission date |
Feb 06, 2006 |
| Last update date |
Feb 07, 2006 |
| Contact name |
Ricardo Dolmetsch |
| E-mail(s) |
ricardo.dolmetsch@stanford.edu
|
| Phone |
650 723 9812
|
| URL |
http://dolmetsch.stanford.edu
|
| Organization name |
Ricardo Dolmetsch
|
| Department |
Neurobiology
|
| Lab |
Dolmetsch
|
| Street address |
299 Campus Drive, D227
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305-5125 |
| Country |
USA |
| |
|
| Platform ID |
GPL891 |
| Series (1) |
| GSE4180 |
CCAT regulated gene expression |
|
