|
| Status |
Public on Jan 31, 2008 |
| Title |
22AVM_200_replicate5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source Name |
200 dpi 22A
|
| Organism(s) |
Mus musculus |
| Characteristics |
infected
|
| Treatment protocol |
C57BL/6 or VM mice were inoculated intracerebrally with 20 µl of 1% brain homogenate from mice infected with scrapie strains 22A, ME7, or 79a.
At the onset of clinical symptoms 8 to 10 mice per sample group were sacrificed and the whole brains used for RNA extraction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
A quantity of 10 µg of total RNA was used as a template for oligo dT primed reverse transcription which incorporated amino allyl -dUTP (Sigma) into the cDNA. The remaining RNA template was hydrolyzed and the cDNA product purified using the QIAquick PCR purification system (Qiagen). PCR products were dried down, then resuspended in 0.1 M Na2CO3 buffer, and incubated for 1 hour in either an Alexa Fluor555 (AF555) or Alexa Fluor647 (AF647) mono-functional reactive dye (Molecular Probes) made up in DMSO. Labelled cDNA was also purified using QIAquick PCR purification columns.
|
| |
|
| Channel 2 |
| Source Name |
Total RNA from pooled 200 day old VM mice
|
| Organism(s) |
Mus musculus |
| Characteristics |
uninfected
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
A quantity of 10 µg of total RNA was used as a template for oligo dT primed reverse transcription which incorporated amino allyl -dUTP (Sigma) into the cDNA. The remaining RNA template was hydrolyzed and the cDNA product purified using the QIAquick PCR purification system (Qiagen). PCR products were dried down, then resuspended in 0.1 M Na2CO3 buffer, and incubated for 1 hour in either an Alexa Fluor555 (AF555) or Alexa Fluor647 (AF647) mono-functional reactive dye (Molecular Probes) made up in DMSO. Labelled cDNA was also purified using QIAquick PCR purification columns.
|
| |
|
| |
| Hybridization protocol |
Labelled cDNA was dried down and resuspended in 60 µl DIG Easy Hyb hybridization buffer (Roche) containing calf thymus DNA (Sigma) and yeast tRNA (Life Technologies). The labelled cDNAs were applied to arrays that contained 11,136 cDNAs from the Brain Molecular Anatomy Project (BMAP). Arrays were constructed in-house as previously described [2]. Hybridization was performed under M Series Lifter Slips (Erie Scientific Company) and the slides incubated in a hybridization chamber at 42oC for 16 hours. Following hybridization lifter slips were removed and the arrays washed a total of 3 times for 5 minutes each in 1 X SSC, 0.2% SDS then 0.1 X SSC, 0.2% SDS pre-warmed to 42C. A final wash in 0.1 X SSC at room temperature was performed and slides spun to dry.
|
| Scan protocol |
Spots were quantitated and background signals removed using Array-Pro® Analyser software (Media Cybernetics, Inc).
|
| Description |
uninfected, age-matched mice used for control
|
| Data processing |
LOWESS normalized, background subtracted VALUE data obtained from log2 of processed Red signal/processed Green signal. GeneTraffic software was used.
|
| |
|
| Submission date |
Jan 24, 2008 |
| Contact name |
stephanie booth |
| E-mail(s) |
Stephanie_Booth@phac-aspc.gc.ca
|
| Organization name |
PHAC
|
| Street address |
1015 Arlington St
|
| City |
Winnipeg |
| ZIP/Postal code |
R3E 2R3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6412 |
| Series (1) |
| GSE10310 |
Comprehensive transcriptional profiling of prion infection in mouse models reveals networks of responsive genes. |
|
