Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of 7G8 cell bank parasites

ABSTRACT: The parasite Plasmodium falciparum is responsible for severe malaria, which remains a major cause of death, particularly in sub-Saharan Africa. The reference strain NF54 (or its subclone 3D7) is commonly used for controlled human malaria infection (CHMI), but recently strains with a different geographic and genomic background have become available for CHMI, including 7G8, which was subcloned from the Brazilian isolate IMTM22 in 1984 (Burkot TR et al. 1984. Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate. Trans R Soc Trop Med Hyg 78 (3):339-41. doi: 10.1016/0035-9203(84)90114-7). To provide the first RNA-seq reference dataset for 7G8 ring stage parasites and validate our qPCR primer set targeting all var gene variants above 6 kb encoded in the 7G8 genome according to current guidelines and best practices, we thawed an aliquot from the Sanaria 7G8 parasite working cell bank (Lot: SAN03-021214 dated 20. February 2014) thawed and cultured for 13 parasite generations at a hematocrit of 5% in human O+ erythrocytes. Parasites were kept synchronous by regular sorbitol treatment. Ring-stage infected erythrocytes were lysed with Trizol, and total RNA was purified using column-based purification (Qiagen RNeasy Mini Mit), including DNase treatment on the column and control for absence of genomic DNA contamination by qPCR. Total RNA samples were cleared of human globin mRNA using magnetic bead isolation technology (GLOBINclear kit). After quality control of RNA samples and optimized preparation of cDNA libraries for AT-rich genomes for Illumina sequencing, RNA-seq was performed at BGI Genomics (Shenzhen, China) on the HiSeq 4000 to generate 100 bp paired-end sequencing reads.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Plasmodium falciparum 7G8

SUBMITTER: Anna Bachmann

PROVIDER: E-MTAB-12157 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Quantitative studies of the P. falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intraerythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade where most expressed genes exhibit a single transcriptional peak. Since proteins represent the decisive business end of gene expression, understanding the correlation between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. While pertinent studies on other organisms show that as little as 20-40% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade where the mRNA levels of most genes change constantly during the IDC. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on spotted oligonucleotide microarrays and 2D-protein gels in combination with DIGE fluorescent dyes. Intriguingly, most proteins are represented by more than one isoform, presumably due to post-translational modifications. Analysis of 366 protein abundance profiles and the corresponding mRNA levels shows that in 67.2% of cases the protein abundance peaks at least 8 hours after the mRNA level peak. While it may be tempting to interpret this as evidence for widespread post-transcriptional gene regulation additional analyses including computer modeling demonstrate that in >60% of these cases the observed protein profiles including the peak lag times could arise as a consequence of the corresponding mRNA levels when simple translation and degradation dynamics are assumed. We further characterize and illustrate these dynamics and show that even human host proteins within the parasite may be subject to similar dynamics as their parasite counterparts. 24 timepoint samples were harvested from a tightly synchronous 6.5 liter biofermenter culture of P. falciparum (Dd2) at 2-hour intervals during one entire intraerythrocytic developmental cycle and compared against a 3D7 RNA reference pool.

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RNA-seq of 7G8 cell bank parasites

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