Computational protocol: Assessment of the relationship between pre-chip and post-chip quality measures for Affymetrix GeneChip expression data

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Protocol publication

[…] A large set of post mortem brain samples (N = 134) that had previously been included in an analysis of gene expression using Human Genome U133A arrays (Affymetrix) were included in the current study (Table ) []. These samples, representing three different brain regions from male and female cases (N = 44) aged 34 to 94, were prepared and processed according to standard protocols (GeneChip® Expression Analysis Protocol, Rev. 2, March 2003, Affymetrix). Briefly, total RNA was extracted using TRIzol (Invitrogen) followed by RNeasy column cleanup (Qiagen) using the manufacturers' protocols. 10 μg total RNA from each sample was used to prepare biotinylated fragmented cRNA, with products from Affymetrix. Arrays were hybridized for 16 h in a 45°C incubator with constant rotation at 60 rpm. Chips were washed and stained on the Fluidics Station 400, and scanned using the GeneArray® 2500, according to the GeneChip® Expression Analysis Protocol, Rev. 2, March 2003 (Affymetrix). All RNA extractions and reactions were prepared using master mixes and batches of 8 and 24 respectively. Arrays were processed in batches of 16. All reactions and array hybridisations were carried out by the same person. The U133A GeneChips came from two manufacturing batches.Gene expression was quantified by robust multi-array analysis (RMA) [] using the affy package [], available as part of the Bioconductor project []. Quality measures were obtained from MAS 5.0 rpt files [], dChip software [], Bioconductor package affyPLM [] and specialised routines for obtaining QC measures from robust regression []. The data are available as part of GEO accession GSE3790. […]

Pipeline specifications

Software tools affy, affyPLM
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Kallmann Syndrome