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Genomic Bioreponse Profiling
While LC/MS represents one of the most powerful
chemical analysis methodologies, there is no single analysis system
that is capable of detecting every type of phytochemical compound.
Hence, a separate and orthogonal methodology is required that
complements the strengths of chemical characterization.
A powerful and novel component of the PhytomicsQC™ platform
is to characterize not only the phytochemical compound pattern but
also the resulting biological response to this collection of phytochemicals.
Using a living human cell line and the genomic
response transcription profile (transcriptome) as a sensitive detector,
one can define an objective, unique subset of the expressed genes
that defines a signature QC pattern for each botanical.
A typical signature set will involve between 20 and 40 genes
that can be developed into a routine plate-based quantitative real
time-PCR (qRT-PCR) assay. Analysis
of the expression profiles of different herbal formulations demonstrate
that the response genes that are differentially expressed form a unique
and quantitative set for each of the botanicals.
This bioresponse gene pattern can be quantified and statistically
compared using the PhytoVeiwerBR™ software analysis tools for the
purpose of QC. In taking this
approach, PhytoCeutica has combined the use of commercially available
gene chips for initial screening with a statistical and informatics
approach for candidate and signature gene set selection, plus real-time
qRT-PCR for confirmation and final assay development. This strategy
is outlined in Figure 2.
Gene expression profiling offers several advantages:
(1) It is a global and general bioassay, capable of integrating the
bioeffects of the entire spectrum of phytochemicals, including small
molecules and macromolecules; (2) gene arrays and qRT-PCR are mature,
robust, and accepted technologies; and (3) each botanical has a unique
and diagnostic gene regulation pattern.
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PhytoCeutica,
Inc. 5 Science Park, Suite 13 New Haven,
CT 06511
Phone: 203-777-3462 Fax: 203-777-3538 E-mail: info@phytoceutica.com |
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> Genomic Bioreponse Profiling _____________________________________________ |
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Figure 2. Workflow for the expression
profiling and unique gene pattern identification of a botanical extract.
Affymetrix microarray chips are used to generate the profile of transcriptional
regulation of over 30,000 genes for the response of the cell line
to a particular botanical. The collection of regulated genes is compared
with the botanical database and statistical algorithms are used to
select a candidate bioresponse
gene that can then be validated and refined using quantitative RT-PCR
to a small, unique set of signature genes. |