Malarial parasite P. falciparum, an apicomplexan protozoan has a 23.3 MB nuclear genome and encodes ~ 5600 transcripts. The genetic diversity of the parasite within and across geographical zones is a challenge to gene expression studies which are essential for understanding of disease process, outcome and developing markers for diagnostics and prognostics. Here, we describe the strategy involved in designing a custom P. falciparum 15K array using the Agilent platform and Genotypic's Right Design methodology to study the transcriptome of Indian field isolates for which genome sequence information is limited. The array contains probes representing genome sequences of two distinct geographical isolates (i.e. 3D7 and HB3) and sub-telomeric var gene sequences of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts through a 244K array experimentation. Array performance for the 15K array, was evaluated and validated using RNA materials from P. falciparum clinical isolates. A large percentage (91%) of the represented transcripts was detected from Indian P. falciparum patient isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. The 15K cross strain P. falciparum array has shown good efficiency in detecting transcripts from P. falciparum parasite samples isolated from patients. The low parasite loads and presence of host RNA makes arrays a preferred platform for gene expression studies over RNA-Seq.
Bibliographical noteKAUST Repository Item: Exported on 2020-10-01
Acknowledgements: We thank all the patients and technical workers for their participation in and support of this project. A.K.S. acknowledges Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), New Delhi, India and Project Assistantship from Department of Biotechnology (DBT), New Delhi, India. P.A.B. acknowledges Basic Scientific Research fellowship from University Grant Commission, New Delhi, India and Project Assistantship from Department of Biotechnology (DBT), New Delhi, India. A.K.D., S.K.K. and D.K.K. acknowledges Department of Biotechnology (DBT), New Delhi, India for the financial support through the grant BT/PR7520/BRB/10/481/2006 and Birla Institute of Technology and Science, Pilani, India and S.P. Medical College, Bikaner, India for providing the required infrastructural facilities during this study. We thank the PlasmoDB team for making available the whole genome expression data for P. falciparum. We also thank Genotypic Technology Pvt. Ltd., Bangalore, India for the microarray hybridization service provided by them. We acknowledge National Institute of Allergy and Infectious Disease (NIAID), National Institute of Health (NIH) funded Genome Sequencing Center for Infectious Diseases at the Broad Institute for making available the HB3 genome sequence and annotation.
ASJC Scopus subject areas
- Molecular Medicine