Bioinformatics and RNA Expression Profiling: Microarray Shared Resource
Using the Affymetrix GeneChip® oligonucleotide array system, the Microarray Shared Resource provides expression profiling of purified RNA samples from a diverse array of organisms including human, mouse and rat.
The Mircroarray Shared Resource provides a full service that includes:
- Consultation of experimental design
- RNA quality check
- cRNA synthesis, labeling and hybridization
- Array scanning
- Data normalization, quality control, and analysis of differential expression
The steps of Full Service:
- Total RNA samples are submitted to the Shared Resource
- The samples are re-purified on Qiagen RNeasy columns
- The samples are analyzed on the Agilent Bioanalyzer and Eppendorf BioPhotometer for RNA integrity (RIN) and concentration
- Samples with RIN values >8.0 are carried forward for cRNA labeling and fragmentation
- The samples are hybridized to the arrays, washed, and scanned to generate .CEL files
- The .CEL files are analyzed for quality assurance using internal Affymetrix parameters and custom signal distribution analyses developed in house by Dr. Chou
- The data are normalized by the MAS5.0 scaling method, Robust MultiChip Average (RMA), or the d-CHIP method, depending on attributes of the experiment or the user’s preference (MAS5.0 is the default)
- The data are intuitively formatted to show probe-gene identities aligned with log2 signal intensity measurements and detection p-values for each gene of each array experiment
- The bioinformatician consults with the user to run an initial analysis of the data, that may, for example, include a statistical analysis whereby Student’s t-test or Wilcoxon rank-sum may be engaged to assess the significance of differential expression of genes between two groups, or more than two groups (eg, ANOVA , Kruskal-Wallis) followed by false discovery correction using the Bejamini & Hochberg method
- Further consultation between the user, the Mircroarray Shared Resource director, and the bioinformatician is encouraged to discuss and pursue further analytical objectives
The GeneChip Process
- Target Preparation: Using the starting sample of total RNA as a template, double stranded cDNA is synthesized, and from that, biotin-labeled cRNA is synthesized. The labeled cRNA is then fragmented.
- Target Hybridization: A hybridization cocktail is prepared using the fragmented cRNA, probe array controls, BSA, and Herring Sperm DNA. The cocktail is then hybridized onto the Genechip for 16 hours.
- Experiment Setup and Fluidics: After hybridization is complete, specified experiment information is defined in the operating software, AGCC. The chips are washed and stained using the fluidics station.
- Probe Array Scan and Data Output: The hybridized chip is scanned using the GeneChip scanner, which outputs a data image file (.dat). The system then computes a cell intensity file (.cel) which averages the intensity of each individual cell on the array. The data collected is then used to form the analysis file (.chp) that contains all of the data output in text format. All data files, along with an excel file of the analysis, is placed on a CD and returned to the client.