Host-pathogen transcriptomics: Trypanosoma cruzi as a model for studying RNA contamination

Transcriptional Studies on Trypanosoma cruzi – Host Cell Interactions: A Complex Puzzle of Variables

“…One of the most common systems to do the parasite propagation is the in vitro infection of Vero (or other types of monkey-derived) cells (Duran-Rehbein et al, 2014). We recently demonstrated that when parasites are grown in a cell line genetically close to the target cells in the infection experiment, false-positive results may occur in the subsequent transcriptomic analysis (Libisch et al, 2020). This contamination happens because, in the trypomastigote RNA purification process, RNA coming from the cell line used to propagate the parasites is also extracted.…”

“…The host cell infection shows considerable heterogeneity at each step of this process, and the specific result may vary with each unique combination of parasite strain, stage (trypomastigotes and/or amastigotes) and host cell type (Epting et al, 2010). Besides, many experimental variables can influence the final result, such as the protocol used to propagate and purify trypomastigotes (Libisch et al, 2020), the interaction time between the parasite and the host cell, the multiplicity of infection (MOI, defined as the ratio of parasites to host cells), the analyzed time, the technology used (qPCR, microarrays or massive sequencing) and the data analysis layout. In addition, it is well known that long lasting T. cruzi cultures can suffer a decrease in the infectivity capacity (Contreras et al, 1998).…”