|
| Status |
Public on Jul 01, 2021 |
| Title |
V63, 10-15h |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NF54
|
| Organism |
Plasmodium falciparum NF54 |
| Characteristics |
strain: NF54
type: mosquito and human passaged parasites
time point: 10-15h
estimated age: 9.5
|
| Treatment protocol |
No treatment performed on samples, just samples of different origin. The NF54 parental line was an in vitro adapted strain always cultured in standard conditions and the volunteer samples came from a previous controlled human malaria infection (CHMI) study in which volunteers were injected with NF54 sporozoites and blood samples were collected 11-14 days later (Gomez-Perez, G. P. et al., 2013. PMID: 26245196). We cultured the volunteer parasites in the same conditions as the NF54 parental line but for the minimum time possible so that their transcriptome wouldn’t be altered by in vitro growth conditions.
|
| Growth protocol |
Parasites were cultured in B+ erythrocytes at a 3 % haematocrit under standard culture conditions in RPMI-based supplemented with 10% 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.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the TRIzol method following the manufacturer instructions.
|
| Label |
Cy5
|
| Label protocol |
Test sample, Cy5; reference pool, Cy3. As previously described (Painter, H. et al., 2013. PMID: 22990780)
|
| |
|
| Channel 2 |
| Source name |
NF54 mixed stage Reference Pool
|
| Organism |
Plasmodium falciparum NF54 |
| Characteristics |
strain: NF54
time point: 3 samples of synchronized cultures covering the whole asexual cycle
|
| Treatment protocol |
No treatment performed on samples, just samples of different origin. The NF54 parental line was an in vitro adapted strain always cultured in standard conditions and the volunteer samples came from a previous controlled human malaria infection (CHMI) study in which volunteers were injected with NF54 sporozoites and blood samples were collected 11-14 days later (Gomez-Perez, G. P. et al., 2013. PMID: 26245196). We cultured the volunteer parasites in the same conditions as the NF54 parental line but for the minimum time possible so that their transcriptome wouldn’t be altered by in vitro growth conditions.
|
| Growth protocol |
Parasites were cultured in B+ erythrocytes at a 3 % haematocrit under standard culture conditions in RPMI-based supplemented with 10% 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.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the TRIzol method following the manufacturer instructions.
|
| 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.
|
| Data processing |
Initial data processing of microarray data, including normalization, was performed using the GE2_1105_Oct12_ACC_2 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. PMID: 22415456). 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 |
Feb 05, 2021 |
| Last update date |
Jul 01, 2021 |
| Contact name |
Anastasia Pickford |
| E-mail(s) |
[email protected]
|
| Organization name |
ISGlobal
|
| Lab |
Malaria Epigenetics
|
| Street address |
Carrer Rosselló 153
|
| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08036 |
| Country |
Spain |
| |
|
| Platform ID |
GPL26985 |
| Series (1) |
| GSE166258 |
Expression patterns of Plasmodium falciparum clonally variant genes at the onset of a blood infection in non-immune humans |
|
