Saint Louis University Gene Chip Facility - Basic Sciences Node
GeneChip is the terminology used by Affymetrix (http://www.affymetrix.com/index.affx) to refer to their proprietary, high-density oligonucleotide array technology. Each GeneChip may contain over 500,000 discrete oligonucleotide sequences or 'probes'. The 'target' nucleic acid of interest may be hybridized to the GeneChip array to determine RNA abundance for expression studies. In the Affymetrix GeneChip platform as many as 40 different oligonucleotide sequences may be used to assay for the abundance of any particular transcript. These probe pair sets contain pairs of exact-match and single nucleotide mismatch sequences that are designed to measure the relative intensity and specificity of the hybridization signal corresponding to their transcript target. Proprietary computer algorithms are then used to compute the relative level of transcript abundance based on the composite hybridization signal from the probe pair set.
Figure 1. Schematic indicating how the abundance of an mRNA species is interrogated by a set of perfect match and mismatch oligonucleotides on the GeneChip.
To perform a 'GeneChip Experiment', 10 ug of purified total cellular RNA from the experimental sample is converted to double stranded cDNA using an oligo-dT primer encoding a T7 RNA polymerase promoter. The purified cDNA is the used in an in vitro transcription reaction to generate biotinylated aRNA. The aRNA is fragmented and hybridized to the GeneChip. The GeneChip is then washed to remove non-specific hybridization and 'stained' with a streptavidin-phycoerythrin conjugate, which allows for fluorescent visualization of the oligonucleotide-aRNA hybrids. The fluorescent signal is quantitated and relative transcript abundance is calculated. A more extensive narrative description of the protocol is available for grant preparation. Alternatively, you may wish to download detailed protocols for preparing samples for GeneChip analysis.
Figure 2. Flow diagram of steps in GeneChip analysis protocol.
What We Do - Services Offered by the GeneChip Facility
Facility users purchase GeneChips through the GeneChip Core facility. A user should contact us 2-3 weeks in advance so that we can order GeneChips for you. You need not submit a PO at the time of ordering, you will be charged for the chips through departmental billing. Please contact the facility to obtain up-to-date prices. Please note that one GeneChip must be used for each sample in your experiment and that GeneChips may be used only once. A complete list of chips available for gene expression profiling is listed with documentation on the Affymetrix web site (http://www.affymetrix.com/products/arrays/index.affx).
Please talk to us first about the gene chips that may be suitable for your experiment.
Generation, Hybridization, and Scanning of Target cRNA
The investigator provides twenty micrograms of total RNA from each experimental sample. Our service includes complete preparation of labeled target cRNA and hybridization cocktail preparation, hybridization to test chip (if requested) and hybridization, washing, scanning, and data storage to CD-ROM for the chips used in the experiment. Data storage includes primary image data and GeneChip data files (accessible by GeneChip software only) as well as a flat text file that may be imported into a spreadsheet such as Microsoft Excel for analysis by Statistical Analysis of Microarrays (SAM) or other software such as the Spotfire analysis package. The facility will review the data to ensure that the experiment was technically successful and provide interactive tutorials and basic guidelines for further data interpretation.
PLEASE NOTE: At present, our facility is covering the cost of reagents and technician’s time in order to lower one barrier to the use of this technology by researchers at the SLU. The cost to investigators is only the cost of the GeneChips used in the experiment. We will continue this policy as long as it is feasible.
An investigator will need at least 10-20 ug of total RNA from each sample. Poly-A enrichment is not necessary and does not improve performance. Total RNA may be prepared using any protocol. For tissue specimens, we recommend an organic extraction protocol (e.g. Trizol, RNAzol, etc.) rather than 'spin-column' methodology. However, if total RNA is isolated by organic extraction, it is absolutely necessary to further purify the RNA using a silica-based spin column methodology (e.g. Qiagen’s RNeasy kit). RNA does not need to be treated with DNase.
Prior to target preparation, an investigator should carefully quantitate the RNA samples. The facility will analyze a small amount of each sample on the Agilent Bioanalyzer in order to confirm the concentration of the sample and assess the quality of the RNA preparation.
Figure 3: Analysis of total RNA sample on Agilent Bioanalyzer chip.
The final RNA concentration must be at least 2 ug/ul to accommodate a small enough volume for cDNA synthesis. The facility will concentrate the samples for you, if necessary. Use nuclease-free water to dissolve the RNA. Do not resuspend your RNA in DEPC-treated water, TE, or other 'RNA protectants', since these may inhibit cDNA synthesis. .
You must submit an Analysis Request Form with each sample. In the first part of this form, you are asked to provide detailed information about the principal investigator and the experiment. In the second part of this form, you should provide the requested information pertaining to each sample. This form is important to avoid any problems in accessing the data from your experiment. All data will eventually be warehoused on a facility server. In summary, please adhere to the following guidelines:
1. RNA must be purified using Qiagen's RNeasy spin columns or other similar methodology.
2. RNA should be quantitated carefully and submitted in nuclease-free water.
All samples should be brought to:
GeneChip Facility- Basic Science Node
Doisy Research Center
Saint Louis University School of Medicine
For additional information, contact:
Michael Green, Ph.D.