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Gene expression profiling of Leishmania (Leishmania) donovani: overcoming technical variation and exploiting biological variation

Published online by Cambridge University Press:  12 October 2007

S. DECUYPERE ,
M. VANAERSCHOT ,
S. RIJAL ,
V. YARDLEY ,
L. MAES ,
S. DE DONCKER ,
F. CHAPPUIS  and
J.-C. DUJARDIN
S. DECUYPERE
Affiliation:
Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of GlasgowG12 8QQ, UK
M. VANAERSCHOT
Affiliation:
Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium Department of Biomedical Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
S. RIJAL
Affiliation:
B.P. Koirala Institute of Health Sciences, Dharan, Nepal
V. YARDLEY
Affiliation:
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
L. MAES
Affiliation:
Laboratory of Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
S. DE DONCKER
Affiliation:
Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium
F. CHAPPUIS
Affiliation:
Department of Community Medicine, Travel and Migration Medicine Unit, Hôpitaux Universitaires de Genève, Rue Micheli-du-Crest 24, CH-1211, Geneva 14, Switzerland
J.-C. DUJARDIN*
Affiliation:
Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium
*
*Corresponding author: Institute of Tropical Medicine, Unit of Molecular Parasitology, Nationalestraat 155, B-2000 Antwerp, Belgium. Tel: +32 3 2476358. Fax: +32 3 2476359. E-mail: jcdujardin@itg.be
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Summary

Gene expression profiling is increasingly used in the field of infectious diseases for characterization of host, pathogen and the nature of their interaction. The purpose of this study was to develop a robust, standardized method for comparative expression profiling and molecular characterization of Leishmania donovani clinical isolates. The limitations and possibilities associated with expression profiling in intracellular amastigotes and promastigotes were assessed through a series of comparative experiments in which technical and biological parameters were scrutinized. On a technical level, our results show that it is essential to use parasite harvesting procedures that involve minimal disturbance of the parasite's environment in order to ‘freeze’ gene expression levels instantly; this is particularly a delicate task for intracellular amastigotes and for specific ‘sensory’ genes. On the biological level, we demonstrate that gene expression levels fluctuate during in vitro development of both intracellular amastigotes and promastigotes. We chose to use expression-curves rather than single, specific, time-point measurements to capture this biological variation. Intracellular amastigote protocols need further refinement, but we describe a first generation tool for high-throughput comparative molecular characterization of patients' isolates, based on the changing expression profiles of promastigotes during in vitro differentiation.

Keywords

Leishmania (Leishmania) donovanistandardizationcomparative expression profiling isolatesvariation gene expression
Type
Research Article
Information
Parasitology , Volume 135 , Issue 2 , February 2008 , pp. 183 - 194
Copyright
Copyright © Cambridge University Press 2007

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