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Focusing on a smaller set of relevant genes
in your DNA microarray studies provides you with more information in less
time. Publicly accessible resources and pre-designed arrays are available to
help you choose the right sets of genes for your research. This article
discusses the concept and advantages of focusing your microarray or RT-PCR
experiments on a pathway-specific set of genes, the resources available to
define a pathway-specific gene list, and how one company has already grouped
genes into pathways to generate Pathway-Centric Tools and Technology™.
1. Why Define Pathway-Specific Genes:
The advancement of microarray technology greatly aids discovery-based
research by allowing investigators to obtain a snapshot of the expression of
an entire genome. Once the relevant genes are discovered, investigators
return to performing hypothesis-driven research in order to better
characterize the biological system at hand. However, due to the need for
expensive equipment and to the high volume of data generated, the
whole-genome techniques remain out of reach for many researchers. Also, many
researchers only wish to focus on one biological pathway or one gene family
and do not necessarily need to examine the entire complement of expressed
genes all at once. A new, more systematic approach to biological research in
this post-genomic era is required.
Organizing genes into pathway-specific lists provides several unique
advantages. The planning of research projects is simplified. The experiment
focuses the research only on the genes or biological pathways of interest.
The execution of research projects is quicker and more efficient. Examining
only roughly one hundred genes instead of thousands at one time yields less
data to examine that is not nearly as overwhelming to interpret. As a result,
you learn more in less time. Also, the overall cost of the experiment becomes
lower.
Low-density or focused microarray experimentation not only serves as an
alternative to high-density work but also serves as a complement.
High-density microarray experiments usually indicate that hundreds to
thousands of genes are differentially regulated by the experimental
conditions. To aid interpretation, most researchers immediately cluster these
genes into their respective biological pathways using on-line database
resources. A pathway-focused microarray provides an alternative platform to
verify that the identified pathways truly play a role in the biological
response in question. The researcher can then examine the entire pathway in a
focused and systematic fashion and in isolation from the rest of the genome.
Thus, low-density microarrays serve as an intermediate step between
high-density arrays and more tedious gene-by-gene microarray data
verification by real-time or conventional RT-PCR.
Combining the current understanding of important biological pathways with the
microarray technologies generates pathway or application focused or specific
research tools. This knowledge-based design merges the benefits of
hypothesis-driven and discovery-based research and allows researchers to
answer more specific questions in a systematic fashion. The continual
revision of the gene content based on new experimental results helps ensure
that these research tools will accelerate, simplify and improve life science
research.
2. Steps Toward Defining Pathway-Specific Genes:
Defining a pathway or application specific set of genes
involves four basic steps. Literature Surveys on the field of study identify
relevant keywords, pathways, and even individual genes. Database Searches
using those terms quickly provide an initial list of specific genes. Other
databases group these gene lists into biological pathways that contain other
genes potentially important to the original research topic. Finally, Expert
Review and User Feedback fine-tunes and optimizes the pathway or
application specific gene list.
Literature Surveys
Comprehensive literature searches on the field of research
help define a list of keywords and other related terms important to the
topic. The search engine known as PubMed,
developed by the National Center for Biotechnology Information (NCBI),
returns lists of scientific publications containing user-defined keywords and
authors. The titles and abstracts of these papers contain other keywords,
biological pathways, and gene families relevant to the field of research
providing other search parameters for PubMed again or other on-line databases
and resources. This process may also reveal a few relevant individual genes.
Collect all of the keywords, pathways, and genes found through the literature
survey.
Database Searches
The Online Mendelian Inheritance in Man™(OMIM™)
resource, a well-curated database of information for individual genes,
returns lists of genes whose entries contain user-defined search terms or
individual genes such as those defined by the literature search. Each entry
in this database represents one gene and contains a brief description as well
as information about cloning, gene function, nomenclature, biochemical
features, gene structure, mapping, molecular genetics, animal models, allelic
variants, and references. Compile the list of genes found from searches of
both OMIM and PubMed using all of the identified keywords.
Lists of genes involved in a subset of biological pathways
or gene families may also be found by browsing corporate web sites such as Biocarta™,
SABiosciences, and others. Biocarta™ displays illustrations of
their featured pathways indicating the relationships between the relevant
gene products. SABiosciences offers DNA microarrays designed for
specific biological pathways. Each array includes a gene list subdivided into
convenient functional gene groupings. The company also offers RT-PCR kits
designed for specific gene families or biological pathways. The individual
product pages provide pre-designed and customizable genes lists for each
available kit. If these web sites contain the same pathways discovered in the
literature or database searches, add the genes to the growing list of focused
genes.
Other publicly accessible search engines and databases
accept individual genes or groups of genes and define all of the pathways to
which they belong. The National Cancer Institute (NCI)
maintains and provides the most comprehensive source of this information, the
Cancer Genome Anatomy Project (CGAP).
Their web site contains a number of interconnected bioinformatic modules
designed for cancer researchers including one used to search "Pathways".
Upon the submission of a list of genes, this search engine returns a table
annotating each gene with terms that correspond to well-defined biological
pathways as defined by other databases including the Gene Ontology (GO)
Consortium, the KEGG PATHWAY
Database, and the previously mentioned resource developed by Biocarta™
The Gene Ontology Consortium consolidates the work of many
collaborating researchers who strive to develop a consistent and universal
set of terms for the description of gene function independent on the genes'
species of origin. The KEGG PATHWAY Database consolidates the most up-to-date
information regarding gene and protein interactions found in regulatory and
metabolic pathways as well as known multi-protein complexes. The annotation
of the pathways in these databases is not necessarily intuitive; therefore,
using CGAP as an entry point to these databases proves very helpful.
Searching the GO or KEGG databases with the terms returned by CGAP provides a
list of even more genes associated with these pathways and the application of
interest.
Other resources, such as HUGO
and PROSITE, focus on gene
families and provide comprehensive lists of genes belonging to those
families. The HUGO Gene Nomenclature Committee maintains the HGNC Gene
Grouping/Family Nomenclature, a simple alphabetical list of gene families and
groupings with hyperlinks to their respective lists of genes. A quick browse
of this web page very quickly reveals whether the resource includes any of
the interesting pathways. The PROSITE web site groups genes into families
based on conserved sequence homology across species in domains of the protein
product. The search engine driving this database accommodates imperfect
matches making the identification of the pathways of interest as well as
other potentially related pathways very easy. Add the resulting lists of
genes from these resources to the growing specific gene list, or feed them
back into the CGAP database to obtain even more pathways and genes.
Expert Review & User Feedback
Even with the best-organized and eminently searchable databases, nothing
beats the human mind and eye when performing a final review of these
pathway-specific gene lists. The designer, a colleague, or another expert in
the relevant field of research should also double-check the gene list for
duplications, inconsistencies, and omissions. The iterative process of
submitting gene lists from one resource to another resource for more genes
introduces redundancies. The on-line resources contain a large amount of
information that the curators update routinely but perhaps not often enough
to keep up-to-date with the literature. An expert in the field who is more
aware of the latest developments could add or remove genes as necessary. In
case reductions need to be made for technical reasons, priorities should be
defined based on the interests of the original designer. Finally, custom
microarray manufacturers can generate pathway- or application-specific
microarrays using the gene list submitted by the designer.
It should be noted that this article and many of the resources discussed
within focus on the more commonly used human and mouse model systems. Despite
the availability of complete genome sequence information for other model
organisms (such as rat, flies and worms), the genes have not yet been as well
curated into pathways as the human or mouse genes. The available resources,
such as FlyBase and WormBase, for these other organisms rely entirely on
submissions made to the GO database. Other ongoing efforts are attempting to
define pathways in other organisms by identifying conserved protein-protein
interactions.
3. Pre-Designed Array Products:
SABiosciences already performs all of these steps
with the release of each Pathway-Centric™
GEArray™ DNA Microarray or MultiGene-12™
RT-PCR Profiling Kit. The company constantly repeats this same process of
literature surveys, database searches, expert review, and user feedback to
generate each product's comprehensive lists of genes. The current collection
of over 100 microarrays representing genes from human, mouse, and rat covers
many if not most research applications. These pre-designed
application-specific arrays save researchers the time and effort in designing
their own specific set of genes. However, the availability of SABiosciences'
Custom Focused DNA Microarray Service allows researchers to obtain
microarrays containing their previously defined list of genes or a
modification of an existing gene list from a cataloged product. The RT-PCR
profiling kits group smaller sets of genes, such as families, together
accommodating even more focused research questions. Investigators also have
the option of modifying the cataloged kits with other genes from the same
gene family or biological pathway to order a kit that better suits their
needs. With these products and services, researchers can immediately reap the
time and cost saving benefits of performing focused or application-specific
gene expression profiling analyses.
Summary of Available Resources Discussed in the Article:
US Government Organizations: National
Center for Biotechnology Information (NCBI) contains multiple search engines
including:
PubMed: Searches for scientific papers by keyword Online Mendelian Inheritance in Man™(OMIM™): Complete,
well-curated, and searchable summary of literature for many human genes. Cancer
Anatomy Genome Project (CGAP): Annotates gene lists to GO, KEGG, and Biocarta
terms
Private Organizations:
Gene Ontology (GO) Consortium: Well-defined biological pathways
Kyoto Encyclopedia of Genes and Genomes (KEGG): Well-defined biological
pathways
HUGO Gene Nomenclature Committee: Well-defined gene families and groupings
PROSITE: Gene families defined by domain with cross-species sequence homology
Corporations: Biocarta:
Illustrations of biological pathways
SABiosciences: Pathway-Centric Tools and Technology™ Microarrays
and RT-PCR kits with pre-designed gene lists
Related Products from SABiosciences:
GEArray™ DNA Microarrays
-
Pathway-Focused Design:
Contains 100-500 well-characterized genes belonging to relevant biological
pathways.
The design reflects the most current understanding of pathways and gene
families.
-
Robust Performance:
Employs the most sensitive proprietary labeling method.
Produces the most reproducible results.
-
Easy to Use & Cost-Effective:
No special equipment required.
Can be used in any laboratory setting.
Priced from $50 per array (US List Price).
MultiGene-12™ RT-PCR Profiling Kits
Profile the expression of eleven closely related genes
in a single analysis. Use a pre-designed gene list or customize one of our
designs. Kits are designed to profile genes: in the same family, in or
responsive to the same biological pathway, useful as disease biomarkers,
with multiple splice variants. Employs a simple assay format with
pre-dispensed primer sets and ready to use PCR master mix. Just mix
template with master mix and transfer to tubes in same strip already
containing primers. Transfer completed reactions directly to agarose gel
wells. Process 8 experiments simultaneously. The pre-validated assay
utilizes primers sets designed with a proprietary, experimentally verified
computer algorithm that yield single PCR products of the predicted size.
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