|
| Status |
Public on Jun 26, 2008 |
| Title |
RNA 106/1(76I_352K) no CQ-treatment, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Pf 106/1(76I_352K), no CQ treatment, synchronized
|
| Organism |
Plasmodium falciparum |
| Characteristics |
parasite line: selected from under 100nM quine (QN)
pfcrt key mutation: K76I_352K
|
| Treatment protocol |
Parasites were synchronized in three steps using successive 5% sorbitol treatments, followed by a Percoll-sorbitol (80%:40%) gradient purification after 24 h and another round of 5% sorbitol treatment, then total RNAs were extracted after another 24 h. Three hours before total RNA extraction, a low dose CQ (IC25, based on previous study) were given to each parasite culture.
|
| Growth protocol |
parasites were maintained in RPMI 1640 medium containing 5% human O+ erythrocytes, 0.5% Albumax, 24 mM sodium bicarbonate and 10 µg/ml gentamycin at 37°C with 5% CO2, 5% O2, and 90% N2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA is extracted following Trizol extraction protocol according to the manufacturer’s instruction. Genomic DNA was extracted from saponin-lysed parasite pellets using Wizard Genomic DNA Purification Kit.
|
| Label |
biotin
|
| Label protocol |
Five microgram total RNA was labeled following Affymetrix standard protocols for eukaryotes using One Cycle Target Labeling and Control Reagent Kit. Ten microgram of genomic DNA was fragmented to an average size of 50–150 bp with DNase I and subsequently end-labeled using terminal deoxynucleotidyl transferase (TdT) and a biotin labeling kit.
|
| |
|
| Hybridization protocol |
After fragmentation, 10 ug of cRNA and DNA were hybridized for 16 hr at 45C on GeneChip Plasmodium/Anopheles Array. GeneChips were washed and stained following the Affymetrix’s EukGE-WS2v5 protocol. Affymetrix 20X hybridization control was used to make the hybridization cocktail.
|
| Scan protocol |
The chips were scanned at 570 nm emission wavelength using Affymetrix scanner 3000.
|
| Description |
RNA 106/1(76I_352K) no CQ-treatment, biological rep3
|
| Data processing |
Image CEL files were processed and normalized using the Robust Multi-array Analysis with correction for GC content of the oligos(GC-RMA).
|
| |
|
| Submission date |
Dec 26, 2007 |
| Last update date |
Jun 26, 2008 |
| Contact name |
Hongying Jiang |
| E-mail(s) |
hojiang@mail.nih.gov
|
| Phone |
3014519033
|
| Fax |
3014022201
|
| Organization name |
NIAID/NIH
|
| Street address |
12735 Twinbrook Parkway
|
| City |
Rockville |
| State/province |
MD |
| ZIP/Postal code |
20852 |
| Country |
USA |
| |
|
| Platform ID |
GPL1321 |
| Series (1) |
| GSE10022 |
Expression and genomic changes after exposing drug-selected mutants to short term CQ treatment in Plasmodium falciparum. |
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