Assay Kits and Custom Services for Research Applications
Monogram utilizes the eTag™ technology to develop and supply assays for multiplexed protein and gene expression analysis to biopharmaceutical researchers. For over two years, eTag assays have been used as an important research tool in the biopharmaceutical laboratories of such companies as GlaxoSmithKline, Pfizer, Sanofi-Aventis and Vertex Pharmaceuticals.
Monogram supplies custom-designed eTag Assays to client specification as well as standard, ready-to-use reagent kits for a wide variety of research applications.
Convenient and Simple Workflow
- Protein-based eTag assays are performed directly on whole cells or cell lysates in microtiter plate wells. No other sample preparation is required.
- eTag-based assays for gene expression are performed directly on cell or tissue lysates in microtiter plate wells, depending on the application. No RNA purification and no other sample preparation is required.
- Convenient mix-wash-read workflow involves minimal handling, and, in the case of gene expression, no need for reverse transcription or polymerase chain reaction (PCR).
Multiplexing for Precise, High Content Information
- The eTag System has the ability to quantitate multiple analytes simultaneously, sensitively, specifically, and accurately — generating superior "high content" results that reflect the actual cell biology. For example, eTag-based gene expression assays can analyze up to 20 different mRNA molecules in the same cell or tissue lysate sample in the same experiment.
- Assays are internally controlled and normalized for variations in cell number, specific proteins, housekeeping genes, total protein content, and total mRNA content.
Flexible Scale
- Applicable in low-, medium-, or selected high-throughput situations, eTag assays are easily automated and scalable.
- eTag assays, which use dramatically less biological sample than other methodologies, can rapidly measure sets of analytes in a few to thousands of samples.
Versatile Applications
- Whether studies involve the analysis of intracellular or extracellular proteins, or transcriptional activity, eTag System is the right platform.
 Elegant Mechanism and Simple Workflow
Protein Analysis Assays
eTag assays can be easily automated using standard liquid handling instrumentation and robotics. Based on the principle of protein proximity, the steps of eTag protein assays involve a simple workflow:
- Small quantities of cells or cell or tissue lysates are added to microtiter well plates.
- The eTag reporter-conjugated reagent (antibody) is added, the mixture is incubated at ambient temperature, and washed.
- The molecular scissor reagent is added.
Any eTag reporter-conjugated antibodies still bound to their specific protein targets after washing are cleaved by the molecular scissors. Then free eTag reporters are released into the solution. The amount of eTag reporters released is proportional to the concentration of target in the sample.
Gene Expression Assays
The eTag Multiplexed Invader® assays1 are able to analyze gene expression in a straightforward manner:
- Small quantities of cell lysate or tissue lysate are added to microtiter well plates. No purification of RNA is required.
- The eTag reporter-conjugated signal probe and the Invader® probe are added, and hybridization to target messenger RNA (mRNA) occurs at 60°C (isothermal conditions).
- Cleavase® reagent is added.
If the eTag reporter-conjugated signal probe and the Invader® probe have hybridized to their complimentary sequences on the target mRNA, a triplex structure of nucleic acid strands is formed. Endonuclease activity of Cleavase® upon the triplex structure results in the cleavage of the 5' end of the target-bound signal probe. Free eTag reporters are released into the solution. The amount of eTag-labeled reporters released is proportional to the concentration of target mRNA in the sample.
Detection and Analysis
After the Cleavase events in protein and gene expression assays, the reaction mixture contains:
- Released eTag reporters (if target was present)
- Uncleaved, unbound eTag reporters
- Cellular proteins, fragments of membranes, and other cellular debris
Separation of released eTag reporters and measurement of fluorescence are accomplished by capillary electrophoresis on high throughput DNA sequencing instruments. Monogram's proprietary eTag Informer™ software quantitates the results and analyzes the data. In the multiplexed assay format, the amount of each released, fluorescent eTag reporter is proportional to the concentration of each target protein or target mRNA molecule in the sample.
The ability to utilize a number of distinctly different eTag reporters in the same assay permits the simultaneous analysis of multiple analytes (e.g. different proteins or mRNA molecules). For example, up to 20 different types of mRNA molecules in the same cell or tissue lysaté sample can be analyzed in the same experiment. Furthermore, because capillary electrophoresis clearly separates the low molecular weight eTag reporters from each other and from the cellular components, the assays have very low background noise, and very precise and sensitive quantitation is achieved.
1 The eTag Multiplex Invader Assay and products incorporate Invader technology and Cleavase enzyme and are licensed for use from Third Wave Technologies, Inc. for multiplexed gene expression applications. FOR RESEARCH USE ONLY; NOT FOR USE IN DIAGNOSTIC PROCEDURES.
eTag Assay System Features
- eTag Multiplex Invader gene expression assays, which involve linear signal amplification, can detect a single copy of mRNA per cell in a sample containing 1000 cells
- eTag-based assays are highly sensitive across a large dynamic range. Both small and large changes in expression of analytes are detected reliably
- As the eTag System can detect and quantitate as few as several hundred receptor proteins per cell, it is almost two orders of magnitude more sensitive than fluorescence-activated cell sorting (FACS)
- Precision and accuracy of eTag assays meet or exceed the performance of ELISAs and Western blots
- Automated separation of released eTag reporters away from background molecules by capillary electrophoresis results in superior sensitivity and increased specificity while eliminating cross-talk problems
- Use of the eTag platform facilitates robust, analyte-independent protocols without a continual need for optimization
- Use of a universal eTag platform results in consistent separation of the same eTag molecules, allowing comparison of data generated in different experiments or laboratories
- The superior sensitivity, accuracy, and reproducibility of the eTag assays enable critical decision-making
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