Dataset Information

Transcriptional profile of Candida glabrata ingested by murine macrophage-like cells (J774A.1) for 2 and 6 hours

ABSTRACT: A set of 5,908 69-70 mers oligonucleotides that correspond to all C. glabrata putative open reading frames and some non-coding sequences was designed and synthesized at Pasteur Institute, France. Each oilgonucleotide was dissolved into 50% DMSO and spotted on UltraGAPsTM glass slides (Corning Incorporated, Acton, MA) in duplicate to generate whole genome microarray for C. glabrata. The microarray was printed at Biotechnology Research Institute/National Research Council of Canada in Montreal. The sequence information of the oligonucleotides and the micorarray setup can be found at platform GPL3922. Using the microarray, the transcriptional profiles of C. glabrata cells that were coincubated with J774A.1 murine Macrophage-like cells for 2 and 6 hrs were examined. At each time point, three biological repeats were done. 20 ug of total RNA from either macrophage-ingested C. glabrata cells (experimental sample) or the medium-alone controls (control sample) was used to synthesize Cy5- or Cy3-labeled cDNA probes with an anchor primer [(dT)18(dA/dG/dC)] in the presence of Cy5- or Cy3-dCTP (PerkinElmer). For each pair of experimental and control samples, dye-swap was performed, that is, two probe mixtures, the Cy5-experimental plus the Cy3-control probes, and conversely, the Cy3-experimental plus the Cy5-control probes were individually hybridized with a single C. glabrata microarray slide. The hybridization and subsequent washing conditions follows the instruction on the manual for UltraGAPsTM glass slides. The hybridized microarrays were scanned with Axon 4000B scanner, and Data were extracted using Axon GenePix program. Log2 (experimental/control) for each spot on the microarray was calculated. From each dye-swap experiment, the Log2 (experimental / control) for each spot were averaged. Finally, 6 Log2 (experimental/control) values (3 repeats × duplicate spots on each slide) were imported into SAM (significance analysis of microarrays) software for statistical analysis. For genes that are considered significantly induced or repressed, the median of false discovery rate (FDR) equals to 0, while the 90% FDR is less than 0.001. Keywords: transcriptional profiling by microarray

ORGANISM(S): Nakaseomyces glabratus

PROVIDER: GSE6058 | GEO | 2006/10/19

SECONDARY ACCESSION(S): PRJNA97739

REPOSITORIES: GEO

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Project description:To identify Six1 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six1wt or VP16-Six1W171R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six1wt infected sample) or Cy5 (AxCAwt VP16-Six1W171R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios. To identify Six4 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six4wt or VP16-Six4W263R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six4wt infected sample) or Cy5 (AxCAwt VP16-Six4W263R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios. Keywords: other

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Transcriptional profile of Candida glabrata ingested by murine macrophage-like cells (J774A.1) for 2 and 6 hours

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