A fast and cost-effective microsampling protocol incorporating reduced animal usage for time-series transcriptomics in rodent malaria parasites.

BACKGROUND: The transcriptional regulation that occurs in malaria parasites during the erythrocytic stages of infection can be studied in vivo with rodent malaria parasites propagated in mice. Time-series transcriptome profiling commonly involves the euthanasia of groups of mice at specific time points followed by the extraction of parasite RNA from whole blood samples. Current methodologies for parasite RNA extraction involve several steps and when multiple time points are profiled, these protocols are laborious, time-consuming, and require the euthanization of large cohorts of mice.

RESULTS: A simplified protocol has been designed for parasite RNA extraction from blood volumes as low as 20 μL (microsamples), serially bled from mice via tail snips and directly lysed with TRIzol reagent. Gene expression data derived from microsampling using RNA-seq were closely matched to those derived from larger volumes of leucocyte-depleted and saponin-treated blood obtained from euthanized mice with high reproducibility between biological replicates. Transcriptome profiling of microsamples taken at different time points during the intra-erythrocytic developmental cycle of the rodent malaria parasite Plasmodium vinckei revealed the transcriptional cascade commonly observed in malaria parasites.

CONCLUSIONS: Microsampling is a quick, robust and cost-efficient approach to sample collection for in vivo time-series transcriptomic studies in rodent malaria parasites.