|
| Status |
Public on Mar 03, 2011 |
| Title |
polyA_RNA_Tdp43_oligo_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Striatum
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 8-10 weeks
treatment: Tdp43 anti-sense oligo
|
| Treatment protocol |
8-10 week old female C57Bl/6 mice were anesthetized with 2.5-3% isofluorane, and a midline incision of approximately 1cm was made on their heads. Using stereotaxic guides, 3 mL of antisense oligonucleotide (ASO) solution targeting Tdp-43 or a control – corresponding to a total of 75 mg or 100 mg ASOs – or saline buffer was injected using a Hamilton syringe directly into their striatum. The incision was closed with sutures and the mice were allowed to recover in their cages. They were monitored for any adverse effects for the next two weeks until they were sacrificed. The striatum and adjacent cortex area were dissected, placed in separate tubes containing 1 mL Trizol (Invitrogen) and were frozen at -80oC until use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of RNA and protein was performed according to the manufacturer’s instructions. In summary, tissues were homogenized in Trizol using a Polytron homogenizer (Company) for 15 sec. Homogenates were then incubated at room temperature for 5min. Chloroform was added to the homogenates (200 ul per 1 mL of Trizol) and tubes were shaken vigorously for 15 sec. After incubation at room temperature for 2-10 min, samples were centrifuged at 12,000 g for 15 min at 4oC. The RNA-containing aqueous phase was transferred to fresh tubes and after the addition of glycogen and isopropanol, RNA was precipitated at -20oC overnight. The next day, samples were centrifuged for 10 mins at maximum speed in a microcentrifuge at 4oC. RNA pellets were washed with 75% Ethanol twice, dried at room temperature and reconstituted in DEPC-treated water.
|
| Label |
biotin
|
| Label protocol |
WT Terminal Labeling Kit, Affymetrix
|
| |
|
| Hybridization protocol |
Hybridization cocktails containing ~5.5 ug of fragmented and labeled DNA target were prepared and applied to GeneChip Human Splice Junction arrays. Hybridization was performed for 16 hours using the Fluidics 450 station.
|
| Scan protocol |
Arrays were scanned using the Affymetrix 3000 7G scanner and GeneChip Operating Software version 1.4 to produce .CEL intensity files. Even though the array was the mjay exon/splicing array, the scanner was set up for MoEx-1_0-st-v1.1sq parameters.
|
| Data processing |
Data processed using Affymetrix package (Affy Power Tools) apt-probeset-summarize. Iter-plier algorithm used to quantify probesets.
|
| |
|
| Submission date |
Feb 16, 2011 |
| Last update date |
Mar 03, 2011 |
| Contact name |
Gene Yeo |
| E-mail(s) |
geneyeo@ucsd.edu
|
| Organization name |
UCSD
|
| Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL13185 |
| Series (2) |
| GSE27371 |
Disrupted processing of long pre-mRNAs and widespread RNA missplicing are components of neuronal vulnerability from loss of nuclear TDP-43 (Affymetrix) |
| GSE27394 |
Disrupted processing of long pre-mRNAs and widespread RNA missplicing are components of neuronal vulnerability from loss of nuclear TDP-43 |
|
