|
| Status |
Public on Feb 13, 2011 |
| Title |
Animal 169 (-seizures) Right/Left |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Animal 169 (-seizures) Right
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: Dentate gyrus
seizure: -
side: right
|
| Treatment protocol |
Adult male rats were given atropine (0.04 mg i.m.), anesthetized with isofluorane 2%, and unilaterally injected with kainic acid (KA; 0.2 µl; 2.0 µg/µl normal saline) in the right posterior CA3 area of hippocampus (AP= -5.6 mm, ML= 4.0 mm, DV= 7.0 mm. A 30ga. needle attached to a Hamilton 1.0 µl syringe was lowered into the injection site and after 5 minutes, one half of the volume was injected. After another 5 minutes, the needle was raised 0.5 mm and the remainder of the solution was injected. After 20 minutes, the needle was withdrawn.
|
| Growth protocol |
Beginning 2 months after kainate injection, video monitoring was performed for all rats in order to detect spontaneous behavioral seizures. After eight months of video monitoring rats were divided for electrophysiological (n=12), microarray (n=10).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ten rats were taken for microarray studies: five with documented behavioral spontaneous seizures and five rats without spontaneous seizures. Rats were anesthetized with isofluorane, brains were quickly removed and sectioned by vibratome into 400 µm slices. Dentate gyri were removed under dissecting microscope and placed in -80C for further microarray experiments. The comparisons were performed between the area of dentate gyri (DG) adjacent to the KA lesion and non-lesioned contralateral side in each animal. RNA extraction was performed using Trizol (Invitrogen). RNA concentration and quality were evaluated using Nanodrop ND-1000 spectrophotometer (NanoDrop) and Agilent 2100 Bioanalyser (Agilent).
|
| Label |
cy5
|
| Label protocol |
We used 100 ng of RNA as the initial starting template, which was labeled with Cy-3 or Cy-5 cytidine -5'-triphosphate (CTP) using the low input fluorescent linear amplification kit (Agilent). The labeled cRNA concentration was verified (Nanodrop) and RNA quality was checked on an Agilent Bioanalyzer (Agilent).
|
| |
|
| Channel 2 |
| Source name |
Animal 169 (-seizures) Left
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: Dentate gyrus
seizure: -
side: left
|
| Treatment protocol |
Adult male rats were given atropine (0.04 mg i.m.), anesthetized with isofluorane 2%, and unilaterally injected with kainic acid (KA; 0.2 µl; 2.0 µg/µl normal saline) in the right posterior CA3 area of hippocampus (AP= -5.6 mm, ML= 4.0 mm, DV= 7.0 mm. A 30ga. needle attached to a Hamilton 1.0 µl syringe was lowered into the injection site and after 5 minutes, one half of the volume was injected. After another 5 minutes, the needle was raised 0.5 mm and the remainder of the solution was injected. After 20 minutes, the needle was withdrawn.
|
| Growth protocol |
Beginning 2 months after kainate injection, video monitoring was performed for all rats in order to detect spontaneous behavioral seizures. After eight months of video monitoring rats were divided for electrophysiological (n=12), microarray (n=10).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ten rats were taken for microarray studies: five with documented behavioral spontaneous seizures and five rats without spontaneous seizures. Rats were anesthetized with isofluorane, brains were quickly removed and sectioned by vibratome into 400 µm slices. Dentate gyri were removed under dissecting microscope and placed in -80C for further microarray experiments. The comparisons were performed between the area of dentate gyri (DG) adjacent to the KA lesion and non-lesioned contralateral side in each animal. RNA extraction was performed using Trizol (Invitrogen). RNA concentration and quality were evaluated using Nanodrop ND-1000 spectrophotometer (NanoDrop) and Agilent 2100 Bioanalyser (Agilent).
|
| Label |
cy3
|
| Label protocol |
We used 100 ng of RNA as the initial starting template, which was labeled with Cy-3 or Cy-5 cytidine -5'-triphosphate (CTP) using the low input fluorescent linear amplification kit (Agilent). The labeled cRNA concentration was verified (Nanodrop) and RNA quality was checked on an Agilent Bioanalyzer (Agilent).
|
| |
|
| |
| Hybridization protocol |
The microarray hybridizations were performed according to manufacturer's protocols.
|
| Scan protocol |
The microarray scanning were performed according to manufacturer's protocols.
|
| Description |
R/L (-Seizures)
|
| Data processing |
We imported the raw data from the Agilent microarrays into R (http://www.r-project.org/). We used available libraries from Bioconductor (http://www.bioconductor.org/) to analyze the two-color microarray designs. We used the linear models package (LIMMA). After the removal of flagged data, we used the default method for background subtraction and used quantile normalization to normalize values between arrays.
|
| |
|
| Submission date |
Feb 02, 2011 |
| Last update date |
Feb 13, 2011 |
| Contact name |
Kellen Winden |
| Organization name |
Boston Children's Hospital
|
| Department |
Neurology
|
| Street address |
300 Longwood Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL2882 |
| Series (2) |
| GSE27015 |
Rat model of MTLE: Animals with epilepsy vs animals without epilepsy (Agilent) |
| GSE27268 |
Rat model of MTLE: Animals with epilepsy vs animals without epilepsy |
|
