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| Status |
Public on Dec 20, 2017 |
| Title |
CA3V_hippocampus_P30_Seizures_4 |
| Sample type |
RNA |
| |
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| Source name |
CA3 ventral hippocampus, P30, Hyperthermic Seizures
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: CA3 ventral hippocampus
time period: P30
group: Hyperthermic seizures
|
| Extracted molecule |
total RNA |
| Extraction protocol |
[experimental protocol] Brain tissue samples were collected from the ventral CA3 hippocampus at 1 (P12), 19 (P30), 49 (P60) and 109 (P120) days after hyperthermic-induced seizures. Brain microdissection was performed as described in (Gorter et al., 2006). Briefly, after decapitation, the temporal lobe and hippocampus were removed by incision at the ventrocaudal part underneath the rhinal fissure, until 5 mm posterior to bregma. Then, the hippocampus was cut into smaller pieces (200-300 uM) and the CA3 region was selected and removed in phosphate buffered saline (PBS) at 4°C under a dissecting microscope. The material obtained from the ventral CA3 region was placed in Eppendorf tubes containing RNA later (Qiagen) for subsequent extraction of total RNA
Total RNA was isolated from CA3 ventral hippocampus samples using Trizol reagent (Life Technologies) and purified using RNeasy Spin Columns (Qiagen, USA). RNA quantity was determined using a Nanovue spectrophotometer (GE Healthcare, USA). The RNA quality was performed using a 2100 Bioanalyzer with an RNA 6000 Nano kit and Ladder (Agilent Technologies, USA), according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the One-Color Microarray-Based Gene Expression Analysis - Low Input RNA (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (Qiagen). RNA concentration and purity was determined by reading the absorbance at 260 and 280 nm on a spectrophotometer (NanoVue, GE Health Care, USA).
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| |
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| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes and hybridized to Agilent Whole Rat Genome Oligo Microarrays (G4122F) for 17 hours at 65°C in a rotating Agilent hybridization oven.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Bundle using one color scan setting for 1x44k array slides.
|
| Description |
Gene expression profiling of CA3 ventral hippocampus at P30
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software version 9.5.3.1 (Agilent) using default parameters (protocol GE1_1105_Oct12 and Grid: 028282_D_F_20150612) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Jul 12, 2016 |
| Last update date |
Dec 20, 2017 |
| Contact name |
Hatylas Azevedo |
| Organization name |
University of São Paulo
|
| Department |
Pediatrics
|
| Street address |
Doutor Arnaldo Avenue, 455
|
| City |
São Paulo |
| State/province |
Sao Paulo |
| ZIP/Postal code |
01246903 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL14746 |
| Series (1) |
| GSE84289 |
Temporal analysis of hippocampal gene co-expression networks in the hyperthermia model of febrile seizures |
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