|
| Status |
Public on Oct 12, 2010 |
| Title |
Time 50% infection 17X |
| Sample type |
RNA |
| |
|
| Source name |
spleen, 50% parasitemia, non-lethal 17X strain
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
tissue: spleen
gender: female
age: 6-8 weeks
|
| Growth protocol |
Parasites from either strain were injected IP into Balb/c mice and daily parasitemia was monitored by Giemsa staining.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Trizol reagent as described in (http://www.medsci.uu.se/klinfarm/arrayplatform/TRIzol_Method%20(2).pdf). The protocol includes lysis of red and white blood cells and DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) or Cyanine-5 (Cy5) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3- (specific activity >10.0 pmol Cy3/ug cRNA) and Cy5-labeled cRNA were fragmented at 60°C for 30 minutes in a reaction volume of 250 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 250 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (GPL2872) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the GenePix 4000 scanner.
|
| Description |
Gene expression spleen at 50% peripheral parasitemia with the non-lethal P. yoelii 17X strain.
Cy3_P50
|
| Data processing |
Bioconductor limma package, using background correction (normexp) and quantiles normalization between arrays for final values. Cy3 and Cy5 channels are normalized values as separate channels for every GPR file.
|
| |
|
| Submission date |
Aug 11, 2009 |
| Last update date |
Oct 12, 2010 |
| Contact name |
Susana Kalko |
| E-mail(s) |
kalko.susana@gmail.com
|
| Organization name |
IDIBAPS
|
| Department |
Bioinformatics Platform
|
| Street address |
c/Rosello 153
|
| City |
Barcelona |
| State/province |
BARCELONA |
| ZIP/Postal code |
08036 |
| Country |
Spain |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE17603 |
Time series of Balb/c spleen infected with Plasmodium yoelii non-lethal vs lethal strains |
|
