|
| Status |
Public on Feb 16, 2018 |
| Title |
TBI+nNos3 virus [nNos3_2] |
| Sample type |
RNA |
| |
|
| Source name |
Hippocampus LCM Neurons, traumatic brain injury, nNos3 knockdown, 2wk, pool from 3 animals
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
received: traumatic brain injury
treated with: nNos3 knockdown siRNA construct
time point: 2 weeks post-treatment
tissue: Hippocampus LCM Neurons
|
| Treatment protocol |
All AAV viral constructs were produced by Vector Biolabs. 3 siRNA sequences targeted to either nNOS or GPX-1 were cloned into the AAV2/1 viral backbone. siRNA oligos were purchased from Ambion and were tested in a neuronal cell culture model to verify gene expression knockdown before providing Vector Biolabs with the sequences for virus production.
Rats received either sham injury, TBI, or TBI + AAV virus (scrambled control virus or nNOS or GPx-1 siRNA constructs) which were stereotaxically injected into the CA1/2 and CA3 regions of the hippocampus. Rats were survived for 15 days, sacrificed, brains were removed and frozen on dry ice. Coronal sections of the brain were collected on glass slides and pyramidal neurons from the CA1/2 and CA3 regions of the hippocampus that expressed the AAV virus were collected by laser capture microdissection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from laser captured hippocampal neurons was isolated and DNase treated prior to microarray analysis.
|
| Label |
Alexa 555
|
| Label protocol |
2 rounds of amplification were performed using the Arcturus RiboAmp HS kit. Biotinylated cRNA target was prepared from the DNA template purified after the second round cDNA synthesis. cRNA quality was verified on an Agilent Bioanalyzer cRNA was fragmented to uniform size and verified on the Bioanalyzer.
|
| |
|
| Hybridization protocol |
10 µg of purified cRNA was fragmented to uniform size and applied to CodeLink Rat Whole Genome Microarrays in hybridization buffer. Arrays were hybridized at 37° C for 18 hrs. in a shaking incubator, and washed at 46° C for 1 hr.
|
| Scan protocol |
Rinsed and dried arrays were scanned with an Axon GenePix 4000B Microarray Scanner at 5 µm resolution.
|
| Data processing |
CodeLink software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies, Santa Clara, CA).
|
| |
|
| Submission date |
Dec 13, 2016 |
| Last update date |
Feb 16, 2018 |
| Contact name |
Michael Falduto |
| E-mail(s) |
[email protected]
|
| Phone |
847-291-9602
|
| Organization name |
GenUs BioSystems, Inc.
|
| Street address |
1808 Janke, Unit M
|
| City |
Northbrook |
| State/province |
IL |
| ZIP/Postal code |
60062 |
| Country |
USA |
| |
|
| Platform ID |
GPL14746 |
| Series (1) |
| GSE92363 |
AAV-mediated RNAi of traumatic brain injury-induced genes in the rat hippocampus |
|
