|
| Status |
Public on Jul 07, 2009 |
| Title |
5362-N2a5822L vs. Mouse Universal Reference Total RNA (Clontech) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
N2a5822L
|
| Organism |
Mus musculus |
| Characteristics |
Neuroblastoma cells stably transfected with Prnp and infected with 22L prion strain
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Qiagen RNeasy kit (Qiagen) then treated with RNase-free DNA I (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
One µg of total RNA N2a5822L was taken and linearly amplified (MessageAMP aRNA kit, Ambion). Two micrograms of amplified RNA were used as a template for oligo(dT)-primed reverse transcription and incorporation of aminoallyl-dUTP into the cDNA, then coupled to cyanine 3 (CyScribe Post-Labelling kit, GE Healthcare).
|
| |
|
| Channel 2 |
| Source name |
Mouse Universal Reference Total RNA, Clontech
|
| Organism |
Mus musculus |
| Characteristics |
Commercial mouse total RNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Commercial mouse total RNA.
|
| Label |
Cy5
|
| Label protocol |
One µg of Mouse Universal Reference Total RNA (Clontech) was taken and linearly amplified (MessageAMP aRNA kit, Ambion). Two micrograms of amplified RNA were used as a template for oligo(dT)-primed reverse transcription and incorporation of aminoallyl-dUTP into the cDNA, then coupled to cyanine 5 (CyScribe Post-Labelling kit, GE Healthcare).
|
| |
|
| |
| Hybridization protocol |
cDNAs coupled to cyanines were hybridised on the mouse developmental array (G4120A, Agilent) in hybridization buffer (Agilent) at 60°C for 17 hours. The slides were then washed 10 min in 6X SSC, 0.005% TritonX-102, then 5 min in 0.1X SSC, 0.005% TritonX-102 on ice.
|
| Scan protocol |
Slides were Slides were dried under filtered nitrogen flow then scanned using a GMS418 scanner (Affymetrix) at adequate laser power.
|
| Description |
To identify genes implied in prion diseases, we chose the N2a58 subline of neuroblastoma cell line. The cells were infected with the 22L prion strain. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen). All RNA samples were treated with RNase-free DNA I (Qiagen). The quality and quantity were initially estimated on the bioanalyzer (Agilent) and quantity of total RNA were confirmed on Nanodrop spectrophotometer. 1 µg of total RNA was amplified using MessageAmp aRNA kit (Ambion). 2 µg of amplified RNA were processed for microarray analysis. Double-strand cDNA was obtained by the reverse transcription using CyScript reverse transcriptase according to the manufacturer specifications (GE Healthcare).
Incorporation of a nucleotide containing an alkyl amino group (AA-dNTP) allowed post-reverse transcription conjugation of the fluorescence dyes. The reaction was blocked by hydroxylamine hydrochloride. Cy-3 and Cy-5 containing probes were combined and purified.
For each sample, the cDNA of the infected or not infected cells were labeled using the green fluorescence dye (Cy3) and the reference cDNA using the red-fluorescent dye (Cy5).
The same amount of cells and reference cDNA was used for the experiment. Cy-3 and Cy-5 containing probes were dried down and resuspended in 100 µl H2O nuclease free and denaturated for 3 min at 98°C. The hybridization was performed on the mouse developmental array (G4120A, Agilent) in hybridization buffer (Agilent) at 60°C for 17 hours. The slides were then washed 10 min in 6X SSC, 0.005% TritonX-102, then 5 min in 0.1X SSC, 0.005% TritonX-102 on ice. Slides were dried under filtered nitrogen flow.
|
| Data processing |
Slides were scanned for both Cy3 and Cy5 with a GMS418 scanner (Affymetrix) and images were processed by ImaGene software (BioDiscovery).
|
| |
|
| Submission date |
Sep 15, 2006 |
| Last update date |
Jul 07, 2008 |
| Contact name |
Jean-Louis Laplanche |
| E-mail(s) |
[email protected]
|
| Phone |
+33149956439
|
| Fax |
+33149958477
|
| Organization name |
Faculté de Pharmacie Université Paris5
|
| Department |
Biologie Cellulaire
|
| Lab |
EA3621
|
| Street address |
4 avenue de l'Observatoire
|
| City |
Paris |
| ZIP/Postal code |
75006 |
| Country |
France |
| |
|
| Platform ID |
GPL922 |
| Series (1) |
| GSE5844 |
Gene expression imprinting of N2a sublines upon infection with the prion strain 22L |
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