Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of 10 individual human cell lines to identify unique genes contributed by individual cell lines to UHRR

ABSTRACT: Total RNA isolated from 10 individual human cell lines were reverse-transcribed to cDNA, labeled with Cy5 and co-hybridized with Cy3-labeled UHRR onto 43,000-spot cDNA microarrays (Stanford University). The data was analyzed using GeneTraffic software. Approximately 6000-8000 spots out of 43,000 (14-18 %) were flagged on each microarray and excluded from further analysis. Spots with hybridization signals in Cy5 channel higher than 1000 and with Cy5/Cy3 ratio greater than 2 were collected and the number of spots with these characteristics in only one cell line was determined.

ORGANISM(S): Homo sapiens

SUBMITTER: Janos Demeter

PROVIDER: E-SMDB-2651 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Universal Reference RNA as a standard for microarray experiments.

Novoradovskaya Natalia N Whitfield Michael L ML Basehore Lee S LS Novoradovsky Alexey A Pesich Robert R Usary Jerry J Karaca Mehmet M Wong Winston K WK Aprelikova Olga O Fero Michael M Perou Charles M CM Botstein David D Braman Jeff J

BMC genomics 20040309 1

<h4>Background</h4>Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed w ...[more]

PMID: 15113400

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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

Dataset Information

Transcription profiling of 10 individual human cell lines to identify unique genes contributed by individual cell lines to UHRR

Publications

Universal Reference RNA as a standard for microarray experiments.

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