|
| Status |
Public on Nov 01, 2020 |
| Title |
Cam3II_R539T_32h_DHA_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Plasmodium falciparum blood stage
|
| Organism |
Plasmodium falciparum |
| Characteristics |
culture: in vitro
Stage: asexual blood stage
strain: Cam3.II
genetic modification: ZFN-edited for K13 mutation
k13 allele: R539T
sampling timepoint: 32h
treatment protocol: 700nM DHA added to 0-3 hpi rings for 6h at start of experiment
experimental replicate: Rep1
|
| Treatment protocol |
0-3 hpi ring stage parasites were treated with 700nM dihydroartemsinin (DHA) or 0.1% DMSO vehicle control for 6h at the start of the experiment and sampled thereafter.
|
| Growth protocol |
Parasites were grown in RPMI1640 media (Gibco, Invitrogen) supplemented with 5% albumax II, 0.2% sodium bicarbonate and 150uM hypoxanthine, and human red blood cells at 3% hematocrit. Cultures were maintained in 3% oxygen and 5% carbon dioxide and kept at 37 degrees celsius.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions and as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211. Amplification of target DNA was carried out as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211, to generate sufficient material for hybridizations against a common RNA reference pool of 3D7 strain using a microarray platform.
|
| Label |
Cy5
|
| Label protocol |
Synthesis of aminoallyl-coupled cDNA by reverse transcription and labeling of samples with cy fluorophore were carried out as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211.
|
| |
|
| Channel 2 |
| Source name |
Plasmodium falciparum reference RNA pool
|
| Organism |
Plasmodium falciparum |
| Characteristics |
culture: in vitro
Stage: asexual blood stage
strain: laboratory strain 3D7
composition: mixture of equal amounts of RNA harvested every 6 hours over the 48-hour parasite's intraerythrocytic life cycle
|
| Treatment protocol |
0-3 hpi ring stage parasites were treated with 700nM dihydroartemsinin (DHA) or 0.1% DMSO vehicle control for 6h at the start of the experiment and sampled thereafter.
|
| Growth protocol |
Parasites were grown in RPMI1640 media (Gibco, Invitrogen) supplemented with 5% albumax II, 0.2% sodium bicarbonate and 150uM hypoxanthine, and human red blood cells at 3% hematocrit. Cultures were maintained in 3% oxygen and 5% carbon dioxide and kept at 37 degrees celsius.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions and as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211. Amplification of target DNA was carried out as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211, to generate sufficient material for hybridizations against a common RNA reference pool of 3D7 strain using a microarray platform.
|
| Label |
Cy3
|
| Label protocol |
Synthesis of aminoallyl-coupled cDNA by reverse transcription and labeling of samples with cy fluorophore were carried out as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211.
|
| |
|
| |
| Hybridization protocol |
Equal amounts of labelled sample and reference cDNA were hybridized on the microarray chip using the Agilent hybridization system. Hybridizations were performed for 20 hours at 65°C in a rotating hybridization oven at 10rpm.
|
| Scan protocol |
Microarray scanning was done using PowerScanner (Tecan, Austria) at 10uM resolution and with autogain PMT adjustments for both channels. Data was acquired using GenePix Pro v6.0 software (Axon Instruments, USA) as described in Mok, S., M. Imwong et al (2011) Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription. BMC Genomics 12: 391.
|
| Data processing |
Features with flag>0 and with median foreground intensity greater than 1.5 fold median background intensity for either channel passed quality control and were retained. Local background correction (normexp) and lowess normalization across all features was applied to generate the normalized log2 gene expression ratios.
|
| |
|
| Submission date |
May 26, 2020 |
| Last update date |
Nov 02, 2020 |
| Contact name |
Sachel Mok |
| E-mail(s) |
[email protected], [email protected]
|
| Organization name |
Nanyang Technological University
|
| Street address |
60 Nanyang Drive
|
| City |
Singapore |
| ZIP/Postal code |
637551 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL18893 |
| Series (1) |
| GSE151189 |
Global RNA expression profiling of k13-edited mutant and isogenic wild-type Plasmodium falciparum malaria parasites during the parasite's intra-erythrocytic developmental cycle and the parasite's response to dihydroartemisinin (DHA). |
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