Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of human peripheral blood mononuclear cells exposed to red blood cells infected with Plasmodium falciparum

ABSTRACT: Peripheral Blood Mononuclear Cells were isolated from three individuals. Cells from these biological replicates were exposed to Red Blood Cells (male donor age 27) infected with Plasmodium falciparum, uninfected Red Blood Cells, or RPMI prior to stimulation with PAM3CSK4 (10g/mL) for 0, 4, and 12 hours. Total RNA was extracted using the Aurum Total RNA Mini Kit (BioRad). Samples were eluted in nuclease-free water and analyzed for concentration and fragment analysis using an Agilent Fragment Analyzer (Agilent). For each sample, 0.5 g of total RNA was used to prepare RNA-seq libraries using the TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat kit and the TruSeq RNA single indexes Set A (Illumina) according to the manufacturer’s instructions. Samples were sequenced at the Bauer Sequencing Core at Harvard University on a HiSeq 2500 (Illumina), using paired-end 75bp sequencing.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Daniel Caffrey

PROVIDER: E-MTAB-9066 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The retention of a nucleus in the mature state of fish red blood cells (RBCs), and the ability to easily collect and manipulate blood in non-terminal experiments, makes it an ideal tissue on which to study the cellular stress response in fish. Through the use of the cGRASP 16K salmonid microarray, we are currently investigating differences in RBC global gene transcription in fish held under control conditions (12C) and exposed to heat stress (one hour at 25C followed by recovery at 12C). Repeated blood sampling (via a dorsal aorta cannula) enables us to examine the individual stress response over time. Samples were taken pre-heat stress (representing individual control) and at 4 and 24 hours post heat stress (representing early and late transcriptional regulation). A total of ~3000 features had significant signal when hybridized with RBC RNA derived targets and cannulation did not have a significant effect on RBC mRNA expression at the investigated time points. Genes involved in the stress response, immune response, and apoptosis were among those showing the highest dysregulation during both early and late transcriptional regulation. Additionally, genes related to the differentiation and development of blood cells were upregulated at the twenty-four hour time point. This study enables a broader understanding of the mechanisms underpinning the stress response in fish and the discovery of novel genes that are regulated in a stress specific manner. Moreover, salmonid transcripts that are consistently dysregulated in blood in response to heat stress are potential candidates of non-lethal biomarkers of exposure to this particular stressor. Two condition experiment, control (11C) and heat stressed fish (one hour at 25C followed by return to 11C). Six fish in each treatment. 3 repeated samples taken from each individual at time 0, 4 hours post heat stress and 24 hours. Common reference design used, with all experimental samples (control or heat stress) combined with reference pool RBC RNA on each array.

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RNA-seq of human peripheral blood mononuclear cells exposed to red blood cells infected with Plasmodium falciparum

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