Ntrol Control Control Control ControlmiRNA pattern A A A A A A A A A A A A A A A A A A A A A A A B A B B A B A B B B B B B B B B B.50 6 10 .50 .20 .10 2 .100 .50 + .100 2 2 2 2 2 2 2 2 10781694 2 2 2 2 2 2 2 2 2 2 232 6 212 541 15 228 13 80 6 6The patient codes are shown on the left. Mn-BC: Buffy coat mini concentration column, number of parasites; “+” means present but not counted; Cell: white cell count in CSF for staging; PCR: presence of parasite DNA; Trypanolysis: positive result from the trypanolysis test; Status: Ser+ 2 positive by CATT; AT: previously treated patient. miR expression pattern: A = more similar to infected, B = more similar to control. doi:10.1371/journal.pone.0067312.tAgilent scanner (SureScan). Data was extracted using Agilent feature extraction software and analyzed with Chipster microarray data analysis software [33]. Each slide has 8 chambers. In each case, three chambers were used for control Title Loaded From File samples and the remaining five were used for individual patient or seropositive samples. All signals were measured relative to the average from thethree controls. Each patient or seropositive sample was analysed once, since there was insufficient material for replicates.qRT-PCRqRT-PCR was carried out to confirm the profiles observed from miRNA expression profiling. To this end, 0.75 mg of total RNAmiRNA in Human 16985061 Sleeping Title Loaded From File Sicknesswere reverse transcribed into cDNA in a total volume of 20 ml using the miScript reverse transcription kit (Qiagen, Hilden, Germany) according to the manufacturers recommendations. Following cDNA synthesis, the resulting cDNA was diluted 10fold before being used for real time PCR. The miScript primer assay for Syber green-based real time PCR (Qiagen) was used for qRT-PCR in a total volume of 12 ml, containing 1 ml of diluted cDNA in a LightCycler 480 system (Roche, Mannheim, Germany). The entire reaction was composed of 40 cycles, consisting of an initial activation step at 95uC for 15 min followed by 40 consecutive cycles of 94uC for 15 sec, 55uC for 30 sec and 70uC for 30 sec for transcript quantification. The U1RNUB6 gene (Qiagen) was used as a standard.targets that went through this filter were subjected to a core analysis in IPA to find out cross relationships and potential downstream effects involving other molecules that could be major players in infection.Statistical AnalysisData analysis was done using Chipster microarray data analysis software [33]. All samples were quintile normalized across chips and filtered according to standard deviation (0.95) and interquartile range. The empirical Baye’s two group t-test (p,0.05) was used to test for differential miRNA expression between different sample groups. The Benjamini-Hochberg correction was applied to all p-value calculations. For linkage clustering, the Pearsons correlation coefficient was calculated. Quantitative real-time (qRT-) PCR was carried out in triplicates for a confirmation of microarray data. Resulting data were expressed as mean 6 standard deviation (SD). All miRNA with a mean difference having a p-value of ,0.05 for a two-sided unpaired student t-test were considered significantly regulated.Gene Expression ProfilingGene expression profiling was performed using the illumina Human Sentrix-12 BeadChip arrays, which contain more than 47,000 probes (Life Technologies, Darmstadt, Germany). Biotinlabeled cDNA samples were prepared according to Illumina’s recommended sample labeling procedure [34]. In brief, 200 ng total RNA was used for.Ntrol Control Control Control ControlmiRNA pattern A A A A A A A A A A A A A A A A A A A A A A A B A B B A B A B B B B B B B B B B.50 6 10 .50 .20 .10 2 .100 .50 + .100 2 2 2 2 2 2 2 2 10781694 2 2 2 2 2 2 2 2 2 2 232 6 212 541 15 228 13 80 6 6The patient codes are shown on the left. Mn-BC: Buffy coat mini concentration column, number of parasites; “+” means present but not counted; Cell: white cell count in CSF for staging; PCR: presence of parasite DNA; Trypanolysis: positive result from the trypanolysis test; Status: Ser+ 2 positive by CATT; AT: previously treated patient. miR expression pattern: A = more similar to infected, B = more similar to control. doi:10.1371/journal.pone.0067312.tAgilent scanner (SureScan). Data was extracted using Agilent feature extraction software and analyzed with Chipster microarray data analysis software [33]. Each slide has 8 chambers. In each case, three chambers were used for control samples and the remaining five were used for individual patient or seropositive samples. All signals were measured relative to the average from thethree controls. Each patient or seropositive sample was analysed once, since there was insufficient material for replicates.qRT-PCRqRT-PCR was carried out to confirm the profiles observed from miRNA expression profiling. To this end, 0.75 mg of total RNAmiRNA in Human 16985061 Sleeping Sicknesswere reverse transcribed into cDNA in a total volume of 20 ml using the miScript reverse transcription kit (Qiagen, Hilden, Germany) according to the manufacturers recommendations. Following cDNA synthesis, the resulting cDNA was diluted 10fold before being used for real time PCR. The miScript primer assay for Syber green-based real time PCR (Qiagen) was used for qRT-PCR in a total volume of 12 ml, containing 1 ml of diluted cDNA in a LightCycler 480 system (Roche, Mannheim, Germany). The entire reaction was composed of 40 cycles, consisting of an initial activation step at 95uC for 15 min followed by 40 consecutive cycles of 94uC for 15 sec, 55uC for 30 sec and 70uC for 30 sec for transcript quantification. The U1RNUB6 gene (Qiagen) was used as a standard.targets that went through this filter were subjected to a core analysis in IPA to find out cross relationships and potential downstream effects involving other molecules that could be major players in infection.Statistical AnalysisData analysis was done using Chipster microarray data analysis software [33]. All samples were quintile normalized across chips and filtered according to standard deviation (0.95) and interquartile range. The empirical Baye’s two group t-test (p,0.05) was used to test for differential miRNA expression between different sample groups. The Benjamini-Hochberg correction was applied to all p-value calculations. For linkage clustering, the Pearsons correlation coefficient was calculated. Quantitative real-time (qRT-) PCR was carried out in triplicates for a confirmation of microarray data. Resulting data were expressed as mean 6 standard deviation (SD). All miRNA with a mean difference having a p-value of ,0.05 for a two-sided unpaired student t-test were considered significantly regulated.Gene Expression ProfilingGene expression profiling was performed using the illumina Human Sentrix-12 BeadChip arrays, which contain more than 47,000 probes (Life Technologies, Darmstadt, Germany). Biotinlabeled cDNA samples were prepared according to Illumina’s recommended sample labeling procedure [34]. In brief, 200 ng total RNA was used for.
