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Tutorials : Jul 1, 2007 ( )
Multiplexed Gene-Expression Assays
Obtaining Accurate Time Course and Dose-response Data and Precise EC50 Values!--h2>
Multiplexed technologies such as High Throughput Genomics’ (HTG; www.htgenomics.com) quantitative nuclease protection assay (qNPA™) for measurement of gene expression address unmet needs in drug discovery and diagnostics. Multiplexed assays mirror what is known about disease and drug mechanisms and offer powerful methods to eliminate false positives and improve quantitative data from whole cell, in vivo tissue, and whole organism experiments. Accurate time course and dose-response data and precise EC50 values can be determined for multiple genes and thousands of samples per day using HTG’s qNPA technology and OMIX Imager. For labs with lower throughput needs, the HTG FireFly Imager (Figure 1) can analyze hundreds of samples a day.
HTG’s multiplexed gene-expression products are based on an array-based nuclease protection assay that does not require the extraction of RNA or PCR (Figure 2), in a flexible microplate format customized on the benchtop by the user to measure any set of 16 genes/well. qNPA is simple, robust, reproducible, accurate, and repeatable. Lysis rather than extraction eliminates quantitative sample-to-sample/gene-to-gene variation due to differences in RNA yield and degradation during extraction. Performed in the presence of nuclease protection probes, the lysis protocol protects target RNA, leading to greater amounts of target remaining in the sample than after extraction.
The number and size of samples is not limiting as qNPA eliminates the variability of reverse transcription and PCR amplification. Measuring invariant housekeeping genes as in-well controls is more reliable than separate controls. The result is a simple, easily automated, reproducible assay with practical sensitivity that is similar to that of PCR, yet delivers better whole-assay reproducibility and repeatability. The ArrayPlate assay is capable of delivering average whole-assay CVs (reproducibility) of <10% for the simultaneous measurement of genes from cells, fresh/frozen/fixed tissues, and whole organisms; excellent dose-response curves; stable time courses and the sample throughput to test as many samples and replicates as necessary; on a flexible, custom microplate platform.
The level of multiplexing and quantitative accuracy provided by the ArrayPlate permits the high-throughput generation of precise EC50s from dose-response curves and is enabling shifts in drug discovery, development, and diagnostics. qNPA can be used to fully exploit the finding from genomics that it is the expression of multiple genes within the transcriptome of transiently expressed genes, and the consequent changes in the proteome, rather than a single gene that causes or is associated with disease. qNPA can be used to exploit the finding that the efficacy and safety of many drugs are related to multiple changes in gene expression. These findings are forcing drug discovery and diagnostics into a new era of multiple target programs and multiplexed measurements.
The HTG FireFly Imager has been specifically designed for use with qNPA kits for directly measuring gene expression on multiple targets simultaneously in a wide range of sample types including formalin-fixed paraffin-embedded tissues without the need for extraction or amplification. The imager allows laboratory researchers in smaller labs to perform studies that previously were not possible due to budget constraints.
The FireFly is a sensitive, charge-coupled device camera (CCD) that can image multiple targets for an entire 96-well plate in about 20 minutes to allow researchers to analyze samples directly at their workstation in the laboratory. The FireFly is ideal for labs where high throughput is not an issue.
Additional benefits include:
• Low background and high resolution enable researchers to discriminate small changes, <20%, in target gene level activity with great confidence, providing quality data to make critical research decisions.
• Ability to read partial plates, allowing increased cost effectiveness in matching plate use to a research budget.
• Simplified, robust optical design that allows high-quality gene-expression experiments on the bench. The HTG FireFly also has a small footprint.
• Proprietary light amplification technology increases sensitivity without the need for a cryogenically cooled CCD chip.
• The software has been designed to allow researchers sophisticated control over the imager. The instrument’s graphic user interface is intuitive and easy to use. This makes it ideal for multi-user environment where scientists can read plates quickly without overly complex instrument controls.
Multiplexed gene-expression assays permit simultaneous rather than sequential measurement, increasing productivity and providing high-content data. The necessity to select a single target is eliminated, reducing hypothesis failure risk and eliminating the months/years of experiments needed to select a single target.
Pursuing drug discovery programs at the level of gene expression rather than at the level of proteins has at least three advantages. It eliminates the time needed to identify the protein targets and develop the required assays; it permits the program to remain multiplexed from start to finish; and combined with the quantitative precision of qNPA, it permits same assay comparison of efficacy, specificity, metabolism, and safety based on quantitative EC50 values.
Enabling gene-based drug discovery requires the capability for high-throughput screening and QSAR optimization, which is achieved by qNPA. Similarly, quantitative multiplexed diagnostic gene-expression assays are expected to provide more accurate understanding of disease state, stage, and subtype, and to monitor drug efficacy to guide effective therapy.
Making the disruptive change to measure a molecular signature (fingerprint) rather than a single target overcomes problems predicated on several generalities. High-throughput screening at the level of whole cells, much less whole organisms, is not typically pursued because of high false positive rates and difficulty differentiating direct/indirect compound effects that may change within an analog series. In vivo data is far less reproducible than in vitro data and less useful for optimization QSAR. Drugs and disease act in the whole organism, not in purified systems, and invariably side effects are discovered as programs move from cell-free to cellular, animal, and then human trials.
For example, agonists and antagonists in purified systems may exhibit mixed activity in cells or in vivo. The objective of therapy is to alter a disease phenotype that results from a molecular phenotype that is typically not measured at each stage of the drug discovery process or diagnostically. A molecular signature can provide both a qualitative measure as well as a quantitative measure of the molecular phenotype. Essentially, use of a molecular signature not only can be used to define the molecular phenotype, but it can also be used to increase the practical sensitivity of the assay.
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