LabAutomation 2006 - SLAS

Where Laboratory Technologies Emerge and Merge
3:30 pm Tuesday, January 24, 2006 Track 3: High-Throughput Technologies Room: Learning Center
Wyndham Palm Springs Hotel
Joseph Monforte
Co-Author(s)
Althea Technologies, Inc.
Gordon Vansant
San Diego, California
Francois Ferre
jmonforte@altheatech.com
Althea Technologies, Inc.
Quantitative Multiplexed Gene Expression Profiling
Global gene expression analysis has proven to be highly informative on the nature and state of cells, including disease progression,
pharmacological response, as well as biological phenomena such as growth and development. In many cases microarray discoveries are
leading to the identification of smaller sets of genes, e.g. 5-50, or signatures, that can provide key information relating to biological state
or response. The eXpress Profiling (XP) gene expression technology utilizes a patented, highly multiplexed PCR approach to quickly and
cost effectively look at the expression of 20-35 genes in a single reaction. Coupled with capillary electrophoresis readout, the method can
efficiently be used to look at focused sets of genes for hundreds or even thousands of samples using very small amounts of total RNA.
4:00 pm Tuesday, January 24, 2006 Track 3: High-Throughput Technologies Room: Learning Center
Wyndham Palm Springs Hotel
Bruce Seligmann
Co-Author(s)
High-Throughput Genomics, Inc.
Costi Sabalos, Ralph Martel
Tucson, Arizona
bseligmann@htgenomics.com
High Throughput Genomics, Inc.
Ron Snyder, George Mandakas
Schering-Plough, Inc.
High Throughput, High Content Gene Expression-Based Target Validation Using Cells,
Fixed or Frozen Tissue, and Whole Organisms to Define the Systems Biology and
Characterize Compound and Stimulus Activity Based on Dose Response EC50 Studies
Whole Genome gene expression methods provide valuable but low-reliability identification of “putative” targets. Extensive validation
is necessary to confirm the importance of these target genes in a disease process or as targets for drug discovery. Target validation
employing PCR is limited by data quality, the lack of sample throughput, the workload that results from using PCR to validate all the
identified putative genes, and inability to accurately measure gene expression from fixed tissues. This latter represents a particular
limitation, considering the existence of vast archives of such tissues in which the gene expression levels induced by disease progression or
drug are essentially “fixed-in-time”. The ArrayPlate quantitative Nuclease Protection Assay (qNPATM) provides a powerful solution and an
independent assay that can build on PCR data (correlation R2>0.9). qNPA fixed tissue results are identical to frozen, making those archives
accessible for target validation. Multiplexed in an open format (measuring 16 genes/well), qNPA delivers high content and high sample
throughput, in an easily automated 96-well microplate format using a simple, robust, lysis-only protocol (identical for cells, frozen/fresh/fixed
tissue, whole organisms) and produces “biochemical” quality data: whole cell assay CV’s of 5% to 10%, repeatable day-to-day within
5%, repeatable lab-to-lab. Precise time course and dose response data (EC50’s) result. Examples: i) How time course precision enables
identification of the “first” Systems Biology events; ii) Genes can be regulated by a drug at characteristically different EC50’s, reflecting
different mechanisms of action of that drug; iii) Clustering of genes into “EC50 signatures” reflecting different “molecular phenotypes”.
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