Design, construction and validation of a Plasmodium vivax microarray for the transcriptome profiling of clinical isolates
2016
Boopathi, Pon Arunachalam | Subudhi, Amit Kumar | Middha, Sheetal | Acharya, Jyoti | Mugasimangalam, Raja Chinnadurai | Kochar, Sanjay Kumar | Kochar, Dhanpat Kumar | Das, Ashis
High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n=14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n=85) present in the arrays showed perfect correlation (r2=0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r≥0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates.
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