Fluorescence‐based assay for accurate measurement of transcriptional activity in trypanosomatid parasites

• Published in: Cytometry. Part A 2018-07, Vol.93 (7), p.727-736
• Main Authors: Hiraiwa, Priscila M., de Aguiar, Alessandra M., Ávila, Andréa R.
• Format: Article
• Language: English
• Subjects: Amanitin; Animals; Assaying; Cell cycle; Flow cytometry; Flow Cytometry - methods; Fluorescence; Gene expression; Growth conditions; high‐content image system; Humans; Identification methods; In vitro methods and tests; in vitro transcription; Parasites; Prophylaxis; Quantitative analysis; Radiolabelling; radiolabel‐free; RNA, Messenger - genetics; Transcription; Transcription, Genetic; Trypanosoma brucei brucei - genetics; Trypanosoma brucei brucei - pathogenicity; trypanosomatids; Trypanosome; Trypanosomiasis, African - genetics; Trypanosomiasis, African - parasitology
• ISSN: 1552-4922; 1552-4930
• DOI: 10.1002/cyto.a.23387

Trypanosomatid parasites are causative agents of neglected human diseases. Their lineage diverged early from the common eukaryotic ancestor, and they evolved singular mechanisms of gene expression that are crucial for their survival. Studies on unusual and essential molecular pathways lead to new drug targets. In this respect, assays to analyze transcriptional activity will provide useful information to identify essential and specific factors. However, the current methods are laborious and do not provide global and accurate measures. For this purpose, a previously reported radiolabeling in vitro nascent mRNA methodology was used to establish an alternative fluorescent‐based assay that is able to precisely quantify nascent mRNA using both flow cytometry and a high‐content image system. The method allowed accurate and global measurements in Trypanosoma brucei, a representative species of trypanosomatid parasites. We obtained data demonstrating that approximately 70% of parasites from a population under normal growth conditions displayed mRNA transcriptional activity, whilst the treatment with α‐amanitin (75 µg/ml) inhibited the polymerase II activity. The adaptation of the method also allowed the analyses of the transcriptional activity during the cell cycle. Therefore, the methodology described herein contributes to obtaining precise measurements of transcriptional rates using multiparametric analysis. This alternative method can facilitate investigations of genetic and biochemical processes in trypanosome parasites and consequently provide additional information related to new treatment or prophylaxis strategies involving these important human parasites. Establishment of a non‐radioactive method for the accurate quantification of transcriptional activity in trypanosomatid cells. The method detect fluorescently labelled nascent RNA in tripanosomatids and can be used for simultaneous multiparametric analysis using flow cytometry or high‐content image system.