Methods to Determine the Transcriptomes of Trypanosomes in Mixtures with Mammalian Cells: The Effects of Parasite Purification and Selective cDNA Amplification

Julius Mulindwa, Abeer Fadda, Clementine Merce, Enoch Matovu, John Enyaru, Christine Clayton

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Patterns of gene expression in cultured Trypanosoma brucei bloodstream and procyclic forms have been extensively characterized, and some comparisons have been made with trypanosomes grown to high parasitaemias in laboratory rodents. We do not know, however, to what extent these transcriptomes resemble those in infected Tsetse flies - or in humans or cattle, where parasitaemias are substantially lower. For clinical and field samples it is difficult to characterize parasite gene expression because of the large excess of host cell RNA. We have here examined two potential solutions to this problem for bloodstream form trypanosomes, assaying transcriptomes by high throughput cDNA sequencing (RNASeq). We first purified the parasites from blood of infected rats. We found that a red blood cell lysis procedure affected the transcriptome substantially more than purification using a DEAE cellulose column, but that too introduced significant distortions and variability. As an alternative, we specifically amplified parasite sequences from a mixture containing a 1000-fold excess of human RNA. We first purified polyadenylated RNA, then made trypanosome-specific cDNA by priming with a spliced leader primer. Finally, the cDNA was amplified using nested primers. The amplification procedure was able to produce samples in which 20% of sequence reads mapped to the trypanosome genome. Synthesis of the second cDNA strand with a spliced leader primer, followed by amplification, is sufficiently reproducible to allow comparison of different samples so long as they are all treated in the same way. However, SL priming distorted the abundances of the cDNA products and definitely cannot be used, by itself, to measure absolute mRNA levels. The amplification method might be suitable for clinical samples with low parasitaemias, and could also be adapted for other Kinetoplastids and to samples from infected vectors.

Original languageEnglish
Article numbere2806
JournalPLoS Neglected Tropical Diseases
Volume8
Issue number4
DOIs
Publication statusPublished - Apr 2014

    Fingerprint

ASJC Scopus subject areas

  • Public Health, Environmental and Occupational Health
  • Infectious Diseases

Cite this