|
| Status |
Public on Mar 22, 2007 |
| Title |
mouse brain ipsilateral LPS.Dmso, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
mouse brain ipsilateral LPS
|
| Organism |
Mus musculus |
| Characteristics |
Strain:C57BL/6
Gender:Male
Age: 60 days
Tissue: brain
Brain dissection: limited at plane anteroposterior +1.5 to -1.5 and dorsoventral -4.0 (Paxino and Franklin, 2001)
|
| Biomaterial provider |
Charles River Canada, St. Constant, Quebec,Canada
|
| Treatment protocol |
C57Bl/6 mouse received an i.p. injection of vehicle (DMSO – 50 microliters) and were submitted to surgery 4 h later. The mice receiving intraparenchymal injections were anesthetized with a mixture of ketamine and xylazine and placed in a stereotaxic apparatus (David Kopf Instruments). The right caudate putamen was reached, using a small cannula (28 gauge; Plastic One) at the coordinates 0.0 mm anteroposterior, -2.0 mm lateral, and -3.0 mm dorsoventral according to a mouse brain atlas [Paxino, G., and Franklin, K. B. J. (2001). The mouse brain in stereotaxic coordinates., 2nd ed (San Diego: Academic Press)]. The animal received an infusion of LPS (from Eschericia coli; serotype O55:B5; Sigma L2880 - 2.5 micrograms - 1 microliter) over 2 min by means of a microinjection 18 pump. Animal was killed 12 h after the intracerebral infusion. The mouse was anesthetized under isofluorane and blood was drawn via cardiac puncture before head decapitation. The brain was removed rapidly from the skull and placed in cold phosphate buffered saline (PBS) solution. A brain region limited at plane anteroposterior +1.5 to -1.5 and dorsoventral -4.0 was dissected, separated in ipsilateral side and quickly immersed in liquid nitrogen. The tissue was stored at -80 oC until RNA extraction was performed.
|
| Growth protocol |
Before and after intracerebral infusion the mouse was kept under standard contidions: 12 h :12 h light : dark cycle; free access to mouse chow; one mouse per cage.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA was isolated using Trizol reagent (Invitrogen) following manufacture’s protocol.
|
| Label |
biotin
|
| Label protocol |
10 micrograms of total RNA was used for cDNA synthesis. Converted to cDNA with SuperScript reverse transcriptase (Gibco-BRL by Invitrogen), using T7-oligo-d(T)24 as a primer. Second-strand synthesis was performed using T4 DNA polymerase and E. Coli DNA ligase followed by blunt ending by T4 polynucleotide kinase. cDNA was isolated by phenol-chloroform extraction using phase lock gels (Brinkmann). cDNA was in vitro transcribed using the T7 BioArray High Yield RNA Transcript Labeling Kit (Enzo Biochem, New York, N.Y.) to produce biotinylated cRNA. Labelled cRNA was isolated using an RNeasy Mini Kit column (Qiagen). Purified cRNA was fragmented to 200–300 mer cRNA using a fragmentation buffer (100 mM potassium acetate-30 mM magnesium acetate-40 mM Tris-acetate, pH 8.1), for 35 min at 94°C. The quality of total RNA, cDNA synthesis, cRNA amplification and cRNA fragmentation was monitored by micro-capillary electrophoresis (Bioanalizer 2100 by Bioanalyser 2100, Agilent Technologies).
|
| |
|
| Hybridization protocol |
cRNA probes were hybridized to an MOE430A Genechip (Affymetrix, Santa Clara, CA). Fifteen micrograms of fragmented cRNA was hybridized for 16 h at 45°C with constant rotation (60 rpm). Microarrays were processed in an Affymetrix GeneChip Fluidic Station 400 protocol EukGE-WS2v4. Staining was made with streptavidin-conjugated phycoerythrin (SAPE) followed by amplification with a biotinylated anti-streptavidin antibody and a second round of SAPE.
|
| Scan protocol |
GeneChips were scanned using the Agilent GeneArray Scanner (Agilent Technologies) ; Pixel Size : 2.5 ; Filter : 570 ;Scanner Type M10.
|
| Description |
mouse brain ipsilateral LPS
|
| Data processing |
The data were analyzed with the RMA summarization algorithm as implemented in Bioconductors package: affy, with deault analysis settings.
|
| |
|
| Submission date |
Dec 12, 2006 |
| Last update date |
Mar 15, 2007 |
| Contact name |
Ariel Chernomoretz |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Buenos Aires
|
| Department |
Physics Department
|
| Street address |
Ciudad Universitaria Pab I
|
| City |
Buenos Aires |
| ZIP/Postal code |
1428 |
| Country |
Argentina |
| |
|
| Platform ID |
GPL339 |
| Series (1) |
| GSE6509 |
Gene Profile of RU486 effect on LPS induced gene expression in CNS. |
|
