Overall Approach to a Genomics Study: Three Phases

High throughput approaches to biological problems such as a microarray functional genomics study are often expensive and inherently produce large volumes of data. It is therefore imperative that the studies be carried out as efficaciously and economically as possible. In order to meet those goals, three important phases of a toxicogenomics study must be successfully completed (Fig. 1).

Phase I is the experimental design of the microarray experiment. The successful completion of phase I is realized when the microarray results are relevant to the scientific questions asked. We encourage at least one meeting with the researchers to discuss the critical issues regarding the experimental design.

Phase II is the carrying out of the microarray experiment itself to generate the raw data, and we carry out preferred protocols covering RNA isolation and RNA quality control assays, RNA amplification, complementary DNA (cDNA) synthesis, cDNA and amplified RNA (aRNA) labeling, microarray slide production, microarray hybridization and wash conditions, and microarray slide scanning and data generation.

Phase III is the analyses of the voluminous amounts of data generated by a typical microarray experiment. The data analysis and data management is carried out by the Dartmouth Biostatistics and Bioinformatics Shared Resource Core directed by Dr. Jason Moore.

Some Useful Information

The spotted DNA sample (known sequence) is referred to as the probe, and the fluorescent-labeled cDNA or cRNA (unknown sequence) is referred to as the target.

To determine experimental variability, it is strongly recommended that a given experiment be carried out at least in triplicate and preferably in quadruplicate. For the same reason, it is also recommended that for one of the slides in the triplicate or for two slides of the quadruplicate, the Cy-3 and Cy-5 labeling are switched for the control and experimental target RNAs.

Experimental Design

When you first begin to consider using microarray technology to answer questions related to your research, the very first step you should take is a discussion of experimental design with Dr. Craig Tomlinson, director of the DGML. He will meet with you to talk about your questions and to help construct an effective design that will help you answer those questions using the microarray facilities. There are many factors that must be considered when using microarrays, including:

There are dozens of experimental variables that may affect microarray experiments, and every researcher's goal should be to eliminate as many of those sources of variation as possible through good experimental design. Some variables to be aware of are:

The DGML can reduce many of those variables by providing full service to researchers. We will begin with quality RNA or DNA (provided by the user) and perform all of the sample preparation and chip processing on our equipment. Therefore, the researcher can be assurred that the majority of detected variation is biological.

A good starting reference for any researcher thinking about using microarray technology is a recent paper in Nature Reviews Genetics: Expression Profiling - Best Practices for Data Generation and Interpretation in Clinical Trials. It gives a good introduction into the best practices for performing a microarray study.

To learn more about how to design your next microarray experiment, please contact the Microarray Core director, Craig Tomlinson, to set up an appointment.

Illumina Deep Sequencing

DNA sequencing has undergone several revolutions in recent decades such that current "next generation" sequencers enable experiments that were, until now, virtually impossible to perform. The Illumina Genome Analyzer II is a next generation "short read" instrument suited for DNA and RNA "resequencing" applications and for metagenomic surveying. Use of the Illumina Genome Analyzer at Dartmouth will enable them to pinpoint genetic mutations and variations associated with phenotypes, determine the genomic locations bound by specific DNA-binding proteins, identify and quantify differences in mRNA expression as a function of environmental conditions, and many other applications. Dartmouth has built all of the computational infrastructure and will provide extensive bioinformatics support for sequence assembly and analysis to enable innovative and high impact science.

See price list for all the available platforms.

For more details about the Illumina deep sequencing platforms, please visit their web site.

Illumina Bead Station

BeadArray Technology : A novel approach to microarrays
Illumina's BeadArray Technology is based on 3-micron silica beads that self assemble in microwells on either of two substrates: fiber optic bundles or planar silica slides. When randomly assembled on one of these two substrates, the beads have a uniform spacing of ~5.7 microns. Each bead is covered with hundreds of thousands of copies of a specific oligonucleotide that act as the capture sequences in one of Illumina's assays.

BeadArray Technology Highlights

Multi-sample array formats
BeadArray technology is deployed on either of two multi-sample array formats for DNA or RNA-analysis applications. With both the 96-sample Array Matrix and the multi-sample BeadChip formats, uniform pits are etched into the surface of each substrate to a depth of approximately 3 microns prior to assembly. Beads are then randomly assembled and held in these microwells by Van der Waals forces and hydrostatic interactions with the walls of the well.

Illumina Bead chips require 50-500ng of total RNA in a maximum volume of 11 uL.

See price list for all the available platforms.

For more details about the Illumina platform, please visit their web site.

Affymetrix Arrays

GeneChip expression arrays enable researchers to simultaneously monitor genome-wide gene expression profiles. This global view helps scientists understand biological mechanisms of complex diseases and processes, and identify new drug targets in ways never before possible. Utilizing gene expression profiles as novel "biomarkers," for conditions such as cancer or lupus, scientists are able to more accurately classify disease, predict clinical progression, and determine likelihoods of treatment success.

The platform utilizes single use microarray chips with one-color (biotin-labeled) detection.

One-cycle (single amplification) Expression requires 5ug of total RNA at a concentration of at least 500ng/uL.

Two-cycle (double amplification) Expression requires at least 10ng in 3uL (50ng in at least 3uL is recommended).

All samples submitted for microarrays must first pass a Quality Check (nanodrop and Agilent Bioanalyzer) request in the DGML.

See price list for all the available platforms.

For more details about the Affymetrix platform, please visit their website.

Agilent Arrays

Agilent's Dual-Mode Gene Expression Analysis Platform gives you the best of both worlds. It delivers both the ease of one-color experimental design and the resolving power of two-color detection - all in a single platform. This unique dual-mode processing capability is built upon a foundation of reliable products that provide a level of quality unmatched in the industry.

Agilent's platform offers either one (single sample Cy3) or two (two samples, one Cy3 and one Cy5) color assays on a glass slide array.

Agilent Expression chips require 50ng-5ug of total RNA in a maximum volume of 8.3uL.

Microarrays - Designed and validated for one-color and two-color processing.

All samples submitted for microarrays must first pass a Quality Check (nanodrop and Agilent Bioanalyzer) request in the DGML.

See price list for all the available platforms.

For more details about the Agilent platform, please visit their website.