Computational protocol: Integrated analysis of the Plasmodium species transcriptome

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Protocol publication

[…] Total RNA was isolated using a standard protocol using trizol/chloroform extraction as described by (). For preparation of the target DNA for microarray hybridization, Switch Mechanism at the 5′ end of Reverse Transcription (SMART) PCR approach was employed (). Thereafter, cDNA was synthesized by using the reverse transcriptase (PowerScript, Clontech BD) for 2 hr at 42 °C. This was followed by PCR amplification with Taq Polymerase (NEB) and the resulting PCR product was purified using MiniElute DNA purification kit (Qiagen). The purified DNA was labeled with fluorescent dye Cy5 (Amersham). A reference pool comprising of equal mass of total RNA samples representing all developmental stages of the parasite was prepared and labeled with Cy3 (Amersham). The microarray hybridization was carried at 65 °C in the automated hybridization station (MAUI, USA). In these two channels competitive hybridizations, RNA from each time point was labeled by Cy5 and hybridized against a reference RNA pool labeled with Cy3. Data acquired were analyzed by GenePix Pro software (Axon Instruments USA). [...] All microarray hybridization spots obtained from all six Plasmodium species were subjected to “normexp” background correction followed by LOWESS (locally weighted scatterplot smoothing) normalization within each array and quantile normalization between arrays using Limma package of R. Log2 ratios of Cy5 over Cy3 intensities were calculated for each spot to represent expression value of a particular probe except those with signal intensity < 1.5 times the background intensity for both Cy5 and Cy3 fluorescence. For each gene, the expression value was estimated as the average of all probes representing it. Overall, 4750 (90% of genes designed on the microarray) P. falciparum genes, 4670 (99%) P. knowlesi genes, 4884 (97%) P. vivax genes, 5486 (88%) P. yoelii genes, 3990 (89%) P. chabaudi genes and 3787 (86%) P. berghei genes display expression profiles with one or zero missing value across each IDC life cycle. These “processed” microarray expression dataset was used for subsequent analysis. The raw and processed microarray data for P. yoelii, P. berghei and P. chabaudi have been deposited into Gene Expression Omnibus (GEO accession number: GSE80015). [...] Ka/Ks ratio is the ratio of the number of nonsynonymous substitutions per non-synonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks). Selective pressure of protein-coding genes between six Plasmodium species was estimated by calculating Ka/Ks ratio for each pair of syntenic orthologs for a total of 2312 orthologs. The syntenic orthologs were aligned using ClustalW () and Ka/Ks were calculated using package ‘seqinr’ of R (). Dendrogram indicating evolution relationship between species was constructed based on the dissimilarity matrix in which the distance of two species was defined by the mode of Ka values. […]

Pipeline specifications

Software tools GenePix Pro, limma, Clustal W, seqinr
Databases GEO
Applications Gene expression microarray analysis, Genome data visualization
Organisms Homo sapiens
Diseases Malaria