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题目:: Transcription profiling of S. cerevisiae GPCR mutants gpr1 and gpa2
ID:
状态:: 发布时间July 8, 2007 , 更新时间 March 27, 2012 , 提交时间 Jan. 16, 2004,
物种:: Saccharomyces cerevisiae
摘要:: Experiment design,Type of experiment: Gene expression profiling analysis of S.cerevisiae W303-1a.,Experimental factor: genetic variation (WT, gpr1 and gpa2 mutant strains).,Number of hybridizations: 6. Single-colour labeling was performed for each sample.,Sample preparation:,Total RNA was prepared using FastRNA Pro Red Kit (BIO 101 Systems) according to the manufacturers' instructions.,Labeling protocol,10 µg of total RNA was reverse transcribed using the SuperScript Choice system for cDNA synthesis (Life Technologies) according to the protocol recommended by Affymetrix. The oligonucleotide used for priming was 5’-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3’ (Genset Oligo). Double-stranded cDNA was cleaned by phenol:chloroform extraction and the aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc.,Hybridization protocol,10 µg of fragmented cRNA were hybridised (45°C, 16 hours). Hybridization was controled by use of the GeneChip™ Eukaryotic Hybridization Control Kit (Affymetrix). Washing and staining was performed in a Fluidics Station 400 (Affymetrix) using the protocol EukGE-WS2v4.,Measurement data, specifications and data processing,Scanning was performed in an Affymetrix GeneChip scanner. Chip analysis was performed using the Affymetrix Microarray Suite v5.,Changes in gene expression were assessed by looking for concordant changes between all replicates using a signed Wilcoxon rank test. The “change” p-value threshold was < 0.025 for increase and > 0.975 for decrease.,Array design,Microarray analysis was performed using YGS98 GeneChips™ oligonucleotide arrays (Affymetrix #900256) representing approximately 6000 ORFs. Sequences and GenBank accession numbers of all probe sets are available at the Affymetrix home page
实验种类:: transcription profiling by array
样本量:: 6
实验设计:
: 无设计数据
数据号:: E-GEOD-970, GDS1485, GSE970
数据状态:: 无法自动分析，您可以尝试手动分析数据。

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