microarray hybridization  (GE Healthcare)

Journal: BMC Genomics

Article Title: Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription

doi: 10.1186/1471-2164-12-391

Figure Lengend Snippet: Transcriptome analyses of the ex vivo cultured P. falciparum parasites . (a) Transcriptomes of the ex-vivo IDC of 6 field isolates (representative set of total 11 transcriptomes generated in this study; Additional file ) from the 3 geographical locations over 48 hour sampling time. The heat maps represent the mean-centered log 2 microarray expression ratios for the P. falciparum genes that were ordered according to the phase calculated by the Fast Fourier Transformation for the reference in vitro IDC transcriptome. (b) Age estimate of the ex vivo culture time points for all 11 field isolates. Each colored box represents age estimate (hpi; shown by numbers outside the circle) of the isolate sample relative to the in vitro reference IDC transcriptome calculated as a best fit Correlation (Spearman rank, see materials and methods). Indicated within each box is the sampling collection time with respect to the first sampling time denoted by 0 h that correspond to the initial sample collection from the infected patients prior to culturing. Sampling times with * represent Spearman Rank correlation value less than 0.35 which indicates a deteriorating synchronicity in the later IDC time points and were excluded from the analysis. Time points included in 3 windows (yellow frame) corresponding to rings at 12-16 hpi, trophozoites at 24 to 28 hpi and schizonts at 32 to 36 hpi were selected for further analysis. (c) Clusters of genes with significant differential expression (p-value < 0.01) between the resistant (grey) and susceptible (orange) parasites at ring (14 hpi), trophozoite (26 hpi) and schizont (34 hpi) stages. Shown are the mean-centered expression log 2 ratios for these genes ranked by the z-score based on the differential expression between the resistant and susceptible isolates. Graphs represent the frequency distribution of the peak abundance time in the IDC transcriptome for each group of genes: over-expressed (red bars), under-expressed (green bars) or no significant change in expression (yellow bars). The grey lines represent the middle of the time IDC interval used for the analysis (e.g. ring, (14 hpi), trophozoite (26 hpi) and schizonts (34 hpi).

Article Snippet: From this, 4 μg of DNA was labeled with fluorescent Cy5 dye and used for the microarray hybridization (GE Amersham, USA).

Techniques: Ex Vivo, Cell Culture, Generated, Sampling, Microarray, Expressing, Transformation Assay, In Vitro, Infection

Journal: BMC Genomics

Article Title: Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription

doi: 10.1186/1471-2164-12-391

Figure Lengend Snippet: Classification of differentially expressed genes across the whole IDC and validation of differential expression of 5 genes by qPCR . (a) Plot of the Ranked Product Score for all 4,015 genes calculated from the geometric mean of the ranked z-scores of the genes in the 3 stages in the generated transcriptome (orange) and in the randomized dataset (green) (for details see material and methods). The graphs represent 4 over (b) and 4 under-expressed (c) genes in the resistant parasites with potential regulatory functions present in the top 5% of each extreme of the rank product distribution, respectively ((a) grey boxes). Data points represent the mean log 2 expression ratios of the genes in the resistant (green triangle), susceptible Lao and Thai (blue diamond), and susceptible Cambodian isolates (CP022, purple circle) across the three selected stage intervals. The data are projected onto the gene expression profiles analyzed by the in vitro IDC transcriptome (red square). Error bars reflect the standard deviation of the log 2 ratios for each data point. (d) Bars represent the log 2 -transformed fold change measured from relative quantification of a resistant versus a sensitive isolate using PFC0965w as a reference control gene in real-time PCR experiments (teal) and plotted alongside the microarray expression ratios (light blue). Error bars reflect the standard deviation of the log 2 ratios over triplicates.

Article Snippet: From this, 4 μg of DNA was labeled with fluorescent Cy5 dye and used for the microarray hybridization (GE Amersham, USA).

Techniques: Expressing, Generated, In Vitro, Standard Deviation, Transformation Assay, Real-time Polymerase Chain Reaction, Microarray

Journal: BMC Genomics

Article Title: Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription

doi: 10.1186/1471-2164-12-391

Figure Lengend Snippet: CGH analysis of artemisinin resistant isolates, Genotyping of isolates and Sequencing of drug-resistant genes to determine haplotypes . (a) The heat map represents hierarchical clustering of CGH signal (against 3d7 genome) for 257 microarray oligonucleotide elements representing 138 genes in 93 CNV regions identified by GADA analysis (see material and methods). (b) Visualization of msp1 , msp2 and glurp nested PCR products of the 6 isolates on a 2.5% agarose gel shows distinctive patterns of product sizes for each clone, except for CP025 and CP037. (c) Tables depict the commonly and rarely found single nucleotide polymorphisms (SNPs) found based on the codon position (top row) and the sequenced bases of the various isolates and corresponding amino acid in brackets. Highlighted in yellow are the codons with mutations compared to wild type 3d7.

Article Snippet: From this, 4 μg of DNA was labeled with fluorescent Cy5 dye and used for the microarray hybridization (GE Amersham, USA).

Techniques: Sequencing, Microarray, Nested PCR, Agarose Gel Electrophoresis