|
| Status |
Public on May 17, 2021 |
| Title |
10E_pfap2-hs 2h post, Control |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
10E pfap2-hs knockout
|
| Organism |
Plasmodium falciparum |
| Characteristics |
strain: 10E_pfap2-hs
treatment: Control
timepoint: 2 h post
estimated age (hpi): 31
|
| Treatment protocol |
Tightly synchronized cultures were exposed to heat shock at 30-35 hpi (10E and 10G) or 33-38 hpi (10E_pfap2-hs). For this, the full incubation chamber was transferred to an incubator at 41.5 ºC for 3 h, and then placed back to 35 ºC. The chamber with the control cultures was always maintained at 35 ºC.
|
| Growth protocol |
Parasites were cultured in B+ erythrocytes at a 3 % haematocrit under standard culture conditions in RPMI-based media containing Albumax II without human serum, in a 5% CO2, 3% O2, balance N2 atmosphere at 35ºC. Tight synchronization (5 h age window) was achieved by Percoll purification followed by sorbitol treatment 5 h later. Given the slower developmental progression of 10E_pfap2-hs, cultures of this parasite line were synchronized to 0-5 hpi 3 h earlier than 10E and 10G cultures, such that at the time of starting HS (in parallel for all lines) all cultures were the same age.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the TRIzol method following the manufacturer intructions.
|
| Label |
Cy5
|
| Label protocol |
Test sample, Cy5; reference pool, Cy3. As previously described (Painter, H. et al., 2013. PMID: 22990780)
|
| |
|
| Channel 2 |
| Source name |
3D7-A mixed stage Reference Pool
|
| Organism |
Plasmodium falciparum |
| Characteristics |
strain: 3D7-A
time-point: 3 samples of synchronized cultures covering the whole asexual cycle.
|
| Treatment protocol |
Tightly synchronized cultures were exposed to heat shock at 30-35 hpi (10E and 10G) or 33-38 hpi (10E_pfap2-hs). For this, the full incubation chamber was transferred to an incubator at 41.5 ºC for 3 h, and then placed back to 35 ºC. The chamber with the control cultures was always maintained at 35 ºC.
|
| Growth protocol |
Parasites were cultured in B+ erythrocytes at a 3 % haematocrit under standard culture conditions in RPMI-based media containing Albumax II without human serum, in a 5% CO2, 3% O2, balance N2 atmosphere at 35ºC. Tight synchronization (5 h age window) was achieved by Percoll purification followed by sorbitol treatment 5 h later. Given the slower developmental progression of 10E_pfap2-hs, cultures of this parasite line were synchronized to 0-5 hpi 3 h earlier than 10E and 10G cultures, such that at the time of starting HS (in parallel for all lines) all cultures were the same age.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the TRIzol method following the manufacturer intructions.
|
| Label |
Cy3
|
| Label protocol |
Test sample, Cy5; reference pool, Cy3. As previously described (Painter, H. et al., 2013. PMID: 22990780)
|
| |
|
| |
| Hybridization protocol |
Hybridization performed as previously described (Painter, H. et al., 2013. PMID: 22990780).
|
| Scan protocol |
Microarrays images were obtained using the Agilent G2505C Microarray Scanner.
|
| Description |
10E_pfap2-hs_2hPost_CTL
|
| Data processing |
Initial data processing of microarray data, including normalization, was performed using the GE2_1105_Oct12 extraction protocol in Agilent Feature Extraction 11.5.1.1 software. The next steps of the analysis were performed using Bioconductor in an R environment (R version 3.5.3). For each individual microarray, we calculated Cy3 and Cy5 background signal as the median of the 100 lowest signal probes for each channel. Probes with both Cy3 and Cy5 signals below three times the array background were excluded (set to NA). Gene level log2(Cy5/Cy3) values and statistical estimation of parasite age were computed as described (Rovira-Graells et al., 2012). Genes with missing values or genes that in all samples had expression values within the lowest 20th percentile of intensity (Cy5 channel) were excluded from downstream analysis, including identification of differentially expressed genes, because differences in genes expressed at near-background levels are of low confidence. To account for the progression delay induced by HS exposure, which can be a major confounder in the downstream analysis, we estimated the age (hours post-invasion, hpi) of each microarray sample by calculating the most likely time post-invasion along the asexual cycle compared to the HB3 transcriptome as reference, using a statistical likelihood method as previously described [Lemieux et al., 2009, PMID: 19376968].
|
| |
|
| Submission date |
Apr 27, 2020 |
| Last update date |
May 18, 2021 |
| Contact name |
Elisabet Tinto-Font |
| E-mail(s) |
elisabet.tinto@gmail.com
|
| Organization name |
ISGlobal
|
| Department |
Malaria
|
| Street address |
c/Rossello 153
|
| City |
Barcelona |
| ZIP/Postal code |
08036 |
| Country |
Spain |
| |
|
| Platform ID |
GPL28456 |
| Series (2) |
| GSE149392 |
P. falciparum heat shock response in a PfAP2-HS-defective population compared to its wild type control. |
| GSE149394 |
Transcriptomic analysis of heat shock resistance in Plasmodium falciparum |
|
