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

Area of Science:

• Molecular Biology
• Parasitology
• Genomics

Background:

• Cellular infection assays are crucial for studying host-pathogen interactions, especially with intracellular microorganisms.
• Propagation of these microorganisms often requires mammalian cell lines, such as Vero cells.
• Potential RNA contamination from host cells can impact transcriptomic study accuracy.

Purpose of the Study:

• To investigate RNA contamination from Vero cells in Trypanosoma cruzi purification.
• To evaluate the impact of this contamination on transcriptomic experiments.
• To develop and recommend an improved purification protocol.

Main Methods:

• Implementation of standard trypanosome purification protocols.
• Detection of Vero cell RNA in trypomastigote extracts and infected human cells using transcriptomic techniques.
• Analysis of RNA-Seq and microarray data for contamination.
• Development and validation of a novel purification method.

Main Results:

• Vero cell-derived RNAs were detected in trypomastigote extracts across multiple purification methods.
• Vero RNA contamination was also found in infected human cells with some protocols.
• Transcriptomic data (microarray, RNA-Seq) showed evidence of spurious results due to contamination.
• The recommended protocol achieved high trypomastigote recovery with no detectable Vero RNA contamination.

Conclusions:

• RNA contamination from cell lines used for pathogen propagation is a significant issue in transcriptomic studies.
• This contamination can lead to false positive results in various transcriptomic analyses.
• An optimized purification protocol is essential for accurate host-pathogen interaction studies and reliable vaccine development.