|
| Status |
Public on Mar 02, 2017 |
| Title |
F12/GDV1-GFP-DDON generation 1 24-32hpi |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RNA extracted from F12/GDV1-GFP-DDON parasites in generation 1 at 24-32hpi
|
| Organism |
Plasmodium falciparum |
| Characteristics |
clone background: F12
genotype/variation: conditional mutant F12/GDV1-GFP-DD
culture conditions: 4nM WR99210; 312nM Shield-1
time point: 24-32 hours post-invasion (hpi)
generation: 1
tissue: whole organism
|
| Growth protocol |
F12/GDV1-GFP-DDOFF parasites were synchronized twice 16 hrs apart to obtain an eight hour growth window (16-24hpi). After re-invasion parasites were synchronised again at 0-8hpi. At 4-12 hpi the culture was split into two populations of which one half was maintained in absence of Shield-1 (F12/GDV1-GFP-DDOFF) and one half was cultured in presence of Shield-1 (F12/GDV1-GFP-DDON). For both populations, total RNA was isolated in the same cell cycle (generation 1) from trophozoites (T1; 28-36 hpi), early schizonts (ES1; 36-44 hpi), late schizonts (LS1; 44-04 hpi), and after re-invasion (generation 2) from early ring stages (ER2; 8-16hpi), late ring stages (LR2; 16-24hpi), trophozoites (T2; 24-32 hpi), early schizonts (ES2; 32-40 hpi).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Ribozol (Amresco) following manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
Labeling was carried out as described in [Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5]
|
| |
|
| Channel 2 |
| Source name |
reference pool RNA
|
| Organism |
Plasmodium falciparum 3D7 |
| Characteristics |
genotype/variation: wild type
time point: 8-48hpi
sample type: cDNA reference pool extracted from wild type parasites (8-48hpi)
tissue: whole organism
|
| Growth protocol |
F12/GDV1-GFP-DDOFF parasites were synchronized twice 16 hrs apart to obtain an eight hour growth window (16-24hpi). After re-invasion parasites were synchronised again at 0-8hpi. At 4-12 hpi the culture was split into two populations of which one half was maintained in absence of Shield-1 (F12/GDV1-GFP-DDOFF) and one half was cultured in presence of Shield-1 (F12/GDV1-GFP-DDON). For both populations, total RNA was isolated in the same cell cycle (generation 1) from trophozoites (T1; 28-36 hpi), early schizonts (ES1; 36-44 hpi), late schizonts (LS1; 44-04 hpi), and after re-invasion (generation 2) from early ring stages (ER2; 8-16hpi), late ring stages (LR2; 16-24hpi), trophozoites (T2; 24-32 hpi), early schizonts (ES2; 32-40 hpi).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Ribozol (Amresco) following manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Labeling was carried out as described in [Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5]
|
| |
|
| |
| Hybridization protocol |
Microarray hybridizations were carried out as described in (Painter HJ, Altenhofen LM, Kafsack BF, Llinás M: Whole-genome analysis of Plasmodium spp. Utilizing a new agilent technologies DNA microarray platform. Methods Mol Biol. 2013;923:213-9). In short, 400ng Cy5-labeled test cDNA and 400ng Cy3-labeled reference pool cDNA were hybridized on a P. falciparum 8x15K Agilent gene expression microarray (GEO platform ID GPL15130) for 16 hours at 65°C in an Agilent hybridisation oven (G2545A).
|
| Scan protocol |
Microarrays were scanned as described in [Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5]. In short, scanning was performed with an Axon GenePix 4000B microarray scanner and associated GenePix Pro v6.0 software (Axon Instruments, Union City, California, USA).
|
| Data processing |
The raw microarray data representing relative transcript abundance ratios between each test sample and the reference pool (Cy5/Cy3 log2 ratios) were subjected to lowess normalization and background filtering as implemented by the Acuity 4.0 program (Molecular Devices). Flagged features and features with either Cy3 or Cy5 intensities lower than two-fold the background were discarded.
|
| |
|
| Submission date |
Mar 01, 2017 |
| Last update date |
Mar 02, 2017 |
| Contact name |
Till Steffen Voss |
| E-mail(s) |
till.voss@swisstph.ch
|
| Phone |
+41612848161
|
| Organization name |
Swiss Tropical and Public Health Institute
|
| Department |
Medical Parasitology and Infection Biology
|
| Lab |
Malaria Gene Regulation
|
| Street address |
Socinstrasse 59
|
| City |
Basel |
| ZIP/Postal code |
4051 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL15130 |
| Series (2) |
| GSE95548 |
Transgenic Plasmodium falciparum blood stage parasites F12/GDV1-GFP-DD: control (-Shield-1;F12/GDV1-GFP-DDOFF) vs GDV1-overexpressing (+Shield-1;F12/GDV1-GFP-DDON) parasites |
| GSE95549 |
Transcriptional response to conditional over-expression of GDV1 in Plasmodium falciparum 3D7 wild type and F12 mutant parasites |
|
