|
| Status |
Public on Jul 14, 2016 |
| Title |
OFF_LS |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RNA extracted from 3D7/TRFGFP-OFF parasites at 40-48hpi
|
| Organism |
Plasmodium falciparum 3D7 |
| Characteristics |
strain: conditional mutant 3D7/TRF-GFP-DD
time point: 40-48 hours post-invasion (hpi)
tissue: whole organism
|
| Growth protocol |
3D7/TRPGFP-ON parasites were synchronized twice 16 hrs apart to obtain an eight hour growth window. At 4-12 hpi the culture was split into two populations, where one third of the culture was maintained in presence of Shield-1 (3D7/TRPGFP-ON) and two thirds were cultured in absence of Shield-1 (3D7/TRPGFP-OFF). At 16-24 hpi both populations were synchronized again. After re-invasion total RNA was isolated from late ring stages (R; 16-24 hpi), trophozoites (T; 24-32 hpi) ), early schizonts (ES; 32-40 hpi) and late schizonts (LS; 40-48 hpi). For 3D7/TRPGFP-OFF parasites a fifth TP was harvested four hrs later (LS2; 44-52 hpi) due to their delay in progression through schizogony
|
| 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 |
strain: wild type strain 3D7
time: 8-48 hours post-invasion (hpi)
tissue: whole organism
|
| Growth protocol |
3D7/TRPGFP-ON parasites were synchronized twice 16 hrs apart to obtain an eight hour growth window. At 4-12 hpi the culture was split into two populations, where one third of the culture was maintained in presence of Shield-1 (3D7/TRPGFP-ON) and two thirds were cultured in absence of Shield-1 (3D7/TRPGFP-OFF). At 16-24 hpi both populations were synchronized again. After re-invasion total RNA was isolated from late ring stages (R; 16-24 hpi), trophozoites (T; 24-32 hpi) ), early schizonts (ES; 32-40 hpi) and late schizonts (LS; 40-48 hpi). For 3D7/TRPGFP-OFF parasites a fifth TP was harvested four hrs later (LS2; 44-52 hpi) due to their delay in progression through schizogony
|
| 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, 250ng Cy5-labeled test cDNA and 250ng Cy3-labeled reference pool cDNA were hybridized on a P. falciparum 8x15K Agilent gene expression microarray 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 |
Jul 13, 2016 |
| Last update date |
Jul 18, 2016 |
| 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 (1) |
| GSE84342 |
Plasmodium falciparum blood stage parasites: control vs PfTRF-depleted parasites |
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