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
GeneChips
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.
Sample Preparation
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. .
Sample Submission
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
Room R413
Doisy Hall
Saint Louis University School of Medicine
For additional information, contact:
Michael Green, Ph.D.
Facility Director
Phone: 977-8897
Email: greenmi@slu.edu
Akbar Siddiqui, B.S.
GeneChip technician
Phone: 977-8899
Email: siddiqui@slu.edu
