|
| Status |
Public on Jul 01, 2011 |
| Title |
Hippocampi Sham replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Hippocampi of sham rats
|
| Organism |
Rattus norvegicus |
| Characteristics |
treatment: sham
sample type: Male albino Wistar rats.
gender: male
strain: albino Wistar
age: 12 weeks old.
tissue: Hippocampi dissected from slices between Bregma levels -2 and -4.
pool: Pooled from three rats.
|
| Treatment protocol |
Rats with electrode placement but without stimulation, handling process similar to stimulated animals
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were conserved in RNA later (Ambion, Austin, TX) for 48 h at 4ºC. Total RNAs were prepared with an RNeasy Lipid Tissue Mini kit according to manufacturer’s protocol (Quiagen, Valencia, CA)
|
| Label |
Cy5
|
| Label protocol |
500 ng of total RNA from each sample were amplified by Oligo-dT-T7 reverse transcription and labeled by in vitro transcription with T7 RNA polymerase in the presence of Cy5-CTP or Cy3-CTP using the Low Input RNA labeling kit (Agilent) and purified using RNAeasy columns (Qiagen, Hilden, Germany)
|
| |
|
| Channel 2 |
| Source name |
Brain tissue of reference
|
| Organism |
Rattus norvegicus |
| Characteristics |
sample type: Male albino Wistar rats.
strain: albino Wistar
age: 12 weeks old.
tissue: Brain pooled tissue consisting of hippocampal, thalamic and cortical brain tissue.
pool: Pooling: naive (n=4), sham-operated (n=5) and stimulated (n=4) rats
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were conserved in RNA later (Ambion, Austin, TX) for 48 h at 4ºC. Total RNAs were prepared with an RNeasy Lipid Tissue Mini kit according to manufacturer’s protocol (Quiagen, Valencia, CA)
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA from each sample were amplified by Oligo-dT-T7 reverse transcription and labeled by in vitro transcription with T7 RNA polymerase in the presence of Cy5-CTP or Cy3-CTP using the Low Input RNA labeling kit (Agilent) and purified using RNAeasy columns (Qiagen, Hilden, Germany)
|
| |
|
| |
| Hybridization protocol |
After fragmentation, 825 ng of labeled cRNA from each of the two samples were co-hybridized in in situ hybridization buffer (Agilent) for 17 h at 65ºC and washed at rt 1 min in 6X SSPE (Saline Sodium Phosphate-EDTA) pH 7.4 + 0.005% Sarcosine, 1 min at rt in Gene Expression WashBuffer 1 (GE WB1 - Agilent), 1 min at 37 ºC with GE WB2 (Agilent).
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner.
Quantified using GenePix 6.0 (Molecular Devices-MDS Analytical Technologies, Silicon Valley, CA). The quality of each individual hybridization was assessed with the Feature Extraction software 10.5 (Agilent).
|
| Description |
Biological replicate 3 of 4. Hippocampi of sham rats versus brain tissue pool of reference.
|
| Data processing |
Raw data was corrected for background noise using the normexp method. Global Lowess normalization was applied (intra-chip normalization). Scaling of the normalized log2 ratios (inter-chip normalization) was applied to assure comparability across samples.
|
| |
|
| Submission date |
Dec 06, 2010 |
| Last update date |
Jul 01, 2011 |
| Contact name |
Gemma Huguet |
| E-mail(s) |
gemma.huguet@udg.edu
|
| Organization name |
Universitat de Girona
|
| Street address |
Campus de Montilivi
|
| City |
Girona |
| ZIP/Postal code |
17071 |
| Country |
Spain |
| |
|
| Platform ID |
GPL11280 |
| Series (1) |
| GSE25860 |
High-frequency stimulation of the ventrolateral thalamus regulates gene expression in hippocampus, motor cortex and caudate-putamen |
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