Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of skeletal muscle from wild type and Six1/ Six4 knock out mice at E10.5 to identify genes under the control of the Six proteins

ABSTRACT: The aim of the experiment was to compare the transcriptome of Six1-/-Six4-/- and control embryos in order to identify genes under the control of Six proteins at E10.5.

E10.5 embryos were eviscerated, head and limbs were discarded, the neural tube was removed, and RNAs were prepared with the remaining axial tissues.

E10.5 RNAs from three SixdKO and two control embryos were hybridized on Affymetrix mouse genome 430A2.0 arrays (Affymetrix, Strasbourg - France).

ORGANISM(S): Mus musculus

SUBMITTER: Claire Niro

PROVIDER: E-MEXP-1849 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 skeletal muscle from wild type and Six1/ Six4 knock out mice at E10.5 to identify genes under the control of the Six proteins

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