|
| Status |
Public on May 27, 2011 |
| Title |
Total Cell Population, GFP +/-, biological rep 1 |
| Sample type |
RNA |
| |
|
| Source name |
TRH-GFP Transfected Hypothalamic Neurons, Total Population GFP+/-
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell type: TRH-GFP Transfected Hypothalamic Neurons, Total Population GFP+/-
tissue: Hypothalamus
strain: Wistar
developmental stage: Embryo
age: E17
|
| Treatment protocol |
Twenty-four hours after seeding, cells were transfected essentially as described before. In general, 8 mg of branched polyethylenimine (PEI) (600-1000 KDa; Fluka) solution was diluted in 10 ml of water, pH adjusted to 6.9 with 0.2 N HCl and the solution filtered (Millipore, 0.22 µm). PEI (30 µl) and plasmid DNA (10 µg) were separately diluted to adjust NaCl to 150 mM in a final volume of 50 µl, vortexed and incubated for 10 min at room temperature; subsequently, the polymer solution was added to the DNA, vortexed-mixed, incubated for 10 min at room temperature followed by the addition of 900 µl of serum-free DMEM. The supplemented DMEM was removed from the culture dishes and the transfection mixture was added. After 3 hours incubation, transfection mix was removed and fresh supplemented DMEM was added.
|
| Growth protocol |
Animals used were Wistar rats raised at the animal facility of the Instituto de Biotecnología, UNAM. Animals were maintained in standard environmental conditions (lights on between 0700-1900 h, temperature 21 ± 2 oC) and received rat chow and tap water ad libitum. Animal care and protocols were approved by the Animal Care and Ethics Committee of the Institute following the guidelines for the use of animals in neuroscience research of the Society for Neuroscience, USA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (10 µg) was extracted from three different cell populations: i) sorted TRH-GFP+ cells (GFP+); ii) TRH-GFP+ and GFP- mixed cells (GFP+/-) passed through the FACS but not sorted, and iii) non transfected cells (NT). To obtain a sufficient amount of RNA for each cell population, the number of independent experiments pooled for the GFP+ sample was higher than for the other samples. Therefore, a pool of six independent experiments was used to prepare total RNA from the GFP+ and three independent experiments for GFP+/- or NT cells.
|
| Label |
Biotin
|
| Label protocol |
RNA was reverse-transcribed into double-stranded cDNA with a T7 promoter-containing primer using SuperScript II Reverse Transcriptase, RNase H, and DNA polymerase (Invitrogen). After precipitation with 5M NH4OAc and ethanol, the cDNA was used as a template in a biotin-labeled in vitro transcription reaction (Enzo BioArray, Affymetrix).
|
| |
|
| Hybridization protocol |
Resulting target cRNA was collected on RNAeasy columns (QIAGEN, Valencia, CA) and then fragmented for hybridization on the microarrays. Biotinylated target cRNA (15 µg) was hybridized to the array and then processed using the Affymetrix GeneChip Fluidics Workstation 400
|
| Scan protocol |
After binding with phycoerythrin-coupled avidin, microarrays were scanned on a Hewlett-Packard Gene Array Scanner (Hewlett-Packard Co., Palo Alto, CA).
|
| Description |
Primary cultures of E17 hypotalamic neurons were transfected with a GFP TRH-promoter drived vector. RNA from total population (GFP+/- cells) was used
|
| Data processing |
Results were analyzed using Affymetrix MAS 5.0 software. Individual microarrays were scaled to produce a mean signal intensity of 125. Iterative comparisons of different microarray datasets were done with MAS 5.0 comparison analysis.
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| |
|
| Submission date |
Apr 07, 2011 |
| Last update date |
May 27, 2011 |
| Contact name |
Leonor Perez-Martinez |
| E-mail(s) |
leonor@ibt.unam.mx
|
| Phone |
52-777-329-1600
|
| Fax |
52-777-317-2388
|
| Organization name |
University of Mexico
|
| Department |
Medicina Molecular y Bioprocesos
|
| Lab |
Neuroimmunobiology
|
| Street address |
Avenida Universidad 2001
|
| City |
Cuernavaca |
| State/province |
Morelos |
| ZIP/Postal code |
62271 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL85 |
| Series (1) |
| GSE28441 |
Transcriptional profiling of fetal hypothalamic TRH neurons |
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