Microarray Help
Normalisation and Display of Microarray Data
CHP files are normalised by averaging values for each cDNA probe across entire datasets. Binary logarithms (log2) of these normalised values are published as heatmaps on the GUDMAP website.
Heatmaps
Heatmaps are a means of displaying the log2 RMA values relative to the median value of each row. Values greater than the median are coloured red whereas values smaller than the median are coloured blue. Values close to the median are coloured black.
Mouseover heatmap to see the log2 RMA values. Alternatively click on "heatmap values", indicated by a red ellipse on the picture, to see log2 RMA values as a table.
Heatmaps are currently available for the following datasets.
- Developing kidney (MOE_430)
- Lower urinary tract (MOE_430)
- Reproductive system (MOE_430)
- Developing kidney (ST_1)
To display heatmaps we use the following formula:
- M (of a probeset in sample i) = RMA (of probeset in sample i) - RMA (median of its value across all samples)
More details on the Affymetrix platforms (MOE_430, ST_1) can be found here.
Genelists from Microarray Analysis
Please view the separate Genelists from Microarray Analysis help page
Detailed Microarray Protocols
Sample and RNA Generation
Most GUDMAP samples for microarray-based gene expression profiling are prepared from timed-pregnant FVB/N females mated with normal or GFP transgenic males. At various times post-coitus, embryos were harvested and initially dissected, further dissected, and sometimes subjected to trypsinization and fluorescence-activated cell sorting (FACS). Micro-dissected, laser-captured or FACS-separated samples were collected in RLT Buffer containing ß-ME, according to manufacturer’s instructions (RNeasy Micro Kit, Qiagen Inc., Valencia, CA) and rapidly frozen in dry ice prior to storage at –80°C for subsequent RNA purification using a spin-column system (RNeasy Micro Kit, Qiagen Inc., Valencia, CA), according to manufacturer’s directions, and stored at –80°C prior to amplification.
RNA Labeling
Purified RNAs are analyzed for concentration and integrity using the Agilent Bioanalyzer RNA 6000 Pico Kit (Agilent Technologies Inc., Santa Clara, CA) and then labeled as appropriate for hybridization to either the Affymetrix Mouse MOE 430 2.0 or Mouse Genes ST 1.0 GeneChip (Affymetrix Inc., Santa Clara, CA) using Epicenter RNA amplification and labeling kits (Epicenter Biotechnologies, Madison, WI) according to manufacturer’s directions with the modification of addition of Full SpectrumTm multistart primers (SBI System Biosciences, Mountain View, CA) per the manufacturer’s directions..
Initial Microarray Data Analysis and the Construction of a Large Gene Expression Matrix File
CEL files were generated from the Affymetrix GeneChip Scanner 3000 7G using Expression Console v1.1.1 (Affymetrix). In a single large batch per microarray type, CEL files were subjected to RMA normalization using Affymetrix CDF probeset definitions. For each array type, the relative expression of each probeset in each sample was compared to that of other samples in the GUDMAP dataset by calculating the ratio of its RMA signal intensity (2**RMA) to that of the median of its values as measured across the entire GUDMAP dataset. Probesets included as part of the core GUDMAP datasets are generally those for which there is no cross-hybridization, had % identity greater than 90% to the mm9 genome reference mouse genome and had RMA intensity value greater than 6.0 in more than one replicate of any sample type. Both the raw RMA and global median normalized expression values are used to form a large-scale expression data matrix that is used for subsequent gene expression profiles, clustering, and gene list generation.
Microrray Analysis Archive
As more microarray samples are submitted to GUDMAP we regularly regenerate the microarray analysis values that are used in the heatmap display. As a consequence the state of the heatmap view will undergo change from time to time. When we update the heatmap values, we archive the previous versions in the Microarray Analysis Archive. Please visit the archive to download previous microarray analysis version as either tab-delimited files or in xls format.
