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QuantiGene Plex assays provide a fast and high-throughput solution for multiplexed gene expression quantitation, allowing the simultaneous measurement of up to 80 genes of interest in a single well of a 96- or 384-well plate. The QuantiGene Plex assay is hybridization-based and incorporates branched DNA (bDNA) technology, which uses signal amplification rather than target amplification for direct measurement of RNA transcripts. The assay is extremely easy to use with a simple ELISA-like workflow, and does not require RNA purification.
Analyze 80 cytokine, chemokine, and growth factor RNA targets simultaneously for efficient immune response profiling, biomarker discovery, and validation.
Browse hundreds of preconfigured and published panels using name, keyword, category, or gene target search. You can also create custom panels.
The QuantiGene Plex Assay utilizes xMAP Luminex beads for multiplexing of 3 to 80 RNA targets followed by branched DNA-based signal amplification. The assay can be performed in 96- or 384-well plates, and signal is detected using a Luminex instrument.
QuantiGene Plex Assays incorporate branched DNA technology and Luminex xMAP technology for accurate multiplex gene expression profiling. Branched DNA assays allow for the direct measurement of RNA transcripts by using signal amplification rather than target amplification.
The QuantiGene Plex Gene Expression Assay is a hybridization-based assay using xMAP Luminex beads and performed in 96- or 384-well plates. The assay is based on direct quantification of the RNA targets for multiplexing of 3 to 80 RNA targets and branched DNA (bDNA) signal amplification technology. On the first day the sample is lysed to release the RNAs and incubated overnight with target specific probe sets and Luminex capture beads. On the second day the signal amplification tree is built via sequential hybridization of Pre-amplifier, Amplifier, and Label Probe. Each amplification unit provides a 400x signal amplification and there are six amplification units per target RNA copy resulting in a 2,400x signal amplification per copy RNA. The signal is detected by using the fluorescent reporter molecule, phycoerythrin, on a Luminex instrument for readout and analysis.
Step 1.Sample preparation: Samples are lysed to release and stabilize RNAs. The RNA assay works with a variety of samples such as: cultured cells, virus, bacteria, plant and animal tissues, FFPE tissues, whole blood and PAXGene blood, or purified RNA.
Step 2. Target hybridization: Overnight hybridization in the 96- or 384-well plates with the target specific probe sets panel (Capture extenders–CEs, Label Extenders–LEs, and Blocking probes).
Step 3. Signal amplification: Signal amplification is achieved using branched DNA (bDNA) technology. First, an individual Pre-Amplifier molecule hybridizes to each pair of Label Extenders. Then, multiple Amplifier molecules hybridize to each Pre-Amplifier. Finally, multiple biotinylated Label Probe oligonucleotides hybridize to each Amplifier.
Step 4. Detection: Addition of streptavidin phycoerythrin (SAPE) generates a signal that is proportional with the amount of target RNA present in the sample. The signal is read using a Luminex instrument.
Step 5. Data Analysis: Analyze data using the Thermo Fisher Connect cloud-based QuantiGene data analysis application from any computer, anywhere. For advanced analysis and visualization, data can be analyzed using the free desktop application software, Applied Biosystems Transcriptome Analysis Console (TAC) software. TAC provides visualization and interpretation using tools such as scatter and volcano plots, hierarchical clustering, and provides link outs to publicly available annotations.
The QuantiGene Plex 384-well assay format enables multiplex gene expression analysis in a high throughput format. The assay uses target-specific probes to capture the RNA of interest and branched DNA technology to amplify fluorescent signals which are read with the FLEXMAP 3D, Luminex platform. Automation and batch processing options for this assay format increase sample throughput and decrease hands-on time while allowing for fast time to results for screening projects.
The QuantiGene Plex 384-well Assay Kit (Cat. No. QP1016) contains all the necessary reagents, buffers, and plates to perform QuantiGene Plex assays in 384-well plates. Design your target-specific QuantiGene Plex panel with our Panel Configuration Tool or contact your local Technical Sales Specialist for more information.
The QuantiGene Plex 384-well assay is designed to increase sample throughput and decrease hands-on time while allowing for fast time to results for screening projects. Below is example data that was generated using the 384-well format using the recommended equipment listed below.
Stimulation of cytokine production in HeLa cells
Cytokine production in cultured HeLa cells was stimulated using a Cell Stimulation Cocktail and monitored for various time points up to 24 hours. In this experiment, a full 80-plex panel was used for screening purposes focused on cytokine, and chemokine biomarkers.
Figure 1. Normalized gene expression of various targets in HeLa cells at different time points post stimulation with a Cell Stimulation cocktail. Y-axis: Average normalized expression; x-axis: mRNA targets; z-axis: time points of stimulation. Only a subset of gene targets tested is being displayed. HeLa cells were cultured in a 384-well format and stimulated with a Cell Stimulation cocktail for various times up to 24h (1h, 2h, 4h, 24h). Unstimulated cells served as baseline control. At the indicated time points, cells were lysed according to the QuantiGene sample prep protocol for cell lysates using liquid handling (Thermo Fisher Platemate). After all samples from all time points were prepared, the QuantiGene Plex 384-well assay was performed. Chart illustrates the normalized gene expression results.
The normalized gene expression of various targets in HeLa cells at different time points post stimulation with a Cell Stimulation cocktail reveals the degree of up- or downregulation. The average normalized expression (y-axis) represents the level of gene induction for the mRNA targets listed on the x-axis, while the time points of stimulation are shown on the z-axis. Of note, for example IL1B, IL8 and TGFB1 show pronounced relative gene expression at various time points of stimulation. While TGFB1 relative expression seems to gradually increase over time, peak expression for IL1B is seen at later time points around 24h. Table 1 shows normalized gene expression and fold change of various targets in HeLa cells at different time points post stimulation with a Cell Stimulation cocktail.
Table 1 and 2. Normalized gene expression and fold change of various targets in HeLa cells at different time points post stimulation with a Cell Stimulation cocktail.
Find instrumentation and equipment to make every step of your QuantiGene Plex 384-well workflow easy. For detailed options and recommendations for batch processing, refer to the QuantiGene Plex 384-well manual or contact your local Technical Sales Specialist.
Workflow step | Recommended equipment | Description | |
---|---|---|---|
Sample incubation and target hybridization | Temperature regulated orbital shaker: MaxQ 4450 Benchtop Orbital Shakers | ![]() | Versatile shaker ideal for incubating a small number of vessels and cell culture and many other applications. |
Plate washes | Magnetic microplate washers | Automated magnetic microplate washer for 384-well plates. | |
Sample incubation | Plate sealer: ALPS 50 V-Manual Heat Sealer | ![]() | Applies consistent, secure, tight seals around individual wells, eliminating sample loss through evaporation and cross contamination between wells. |
Sample and plate additions | Versette Automated Liquid Handler | ![]() | Compact liquid handler featuring user-friendly programming, reliable performance, and a choice of 96- or 384-channel pipetting heads. |
Multiplex detection | Luminex FLEXMAP 3D Instrument System | ![]() | Most advanced and multifunctional multiplex unit for simultaneous measurement of up to 80 tests in a single reaction volume. |
Parameter | 96-well | 384-well | |
---|---|---|---|
Product specification | Sensitivity (Limit of detection) | 1,000–2,000 transcripts/assay well | 2,000–4,000 transcripts/assay well |
Assay variation (C.V.) | ≤15% | ≤20% | |
Linearity | ≥3 logarithmic units | ||
Plex level | 3–80 | 3–80 | |
Samples | Cultured cells, bacteria, whole blood, PAXgene blood or dried blood spots, fresh/frozen tissues (animal or plant), FFPE samples, purified RNA | ||
Compatible Luminex instrumentation | MAGPIX, Luminex 200, FLEXMAP 3D, INTELLIFLEX | FLEXMAP 3D, INTELLIFLEX | |
Assay volumes | Target probe hybridization | 100 µL | 70 µL |
Pre-amp hybridization | 100 µL | 30 µL | |
Amp hybridization | 100 µL | 30 µL | |
Label probe hybridization | 100 µL | 30 µL | |
SAPE | 100 µL | 30 µL |
User manuals for use with assay kits QP1013, QP1014, QP1015 and QP1016
QuantiGene sample processing
The QuantiGene Plex Assay utilizes xMAP Luminex beads for multiplexing of 3 to 80 RNA targets followed by branched DNA-based signal amplification. The assay can be performed in 96- or 384-well plates, and signal is detected using a Luminex instrument.
QuantiGene Plex Assays incorporate branched DNA technology and Luminex xMAP technology for accurate multiplex gene expression profiling. Branched DNA assays allow for the direct measurement of RNA transcripts by using signal amplification rather than target amplification.
The QuantiGene Plex Gene Expression Assay is a hybridization-based assay using xMAP Luminex beads and performed in 96- or 384-well plates. The assay is based on direct quantification of the RNA targets for multiplexing of 3 to 80 RNA targets and branched DNA (bDNA) signal amplification technology. On the first day the sample is lysed to release the RNAs and incubated overnight with target specific probe sets and Luminex capture beads. On the second day the signal amplification tree is built via sequential hybridization of Pre-amplifier, Amplifier, and Label Probe. Each amplification unit provides a 400x signal amplification and there are six amplification units per target RNA copy resulting in a 2,400x signal amplification per copy RNA. The signal is detected by using the fluorescent reporter molecule, phycoerythrin, on a Luminex instrument for readout and analysis.
Step 1.Sample preparation: Samples are lysed to release and stabilize RNAs. The RNA assay works with a variety of samples such as: cultured cells, virus, bacteria, plant and animal tissues, FFPE tissues, whole blood and PAXGene blood, or purified RNA.
Step 2. Target hybridization: Overnight hybridization in the 96- or 384-well plates with the target specific probe sets panel (Capture extenders–CEs, Label Extenders–LEs, and Blocking probes).
Step 3. Signal amplification: Signal amplification is achieved using branched DNA (bDNA) technology. First, an individual Pre-Amplifier molecule hybridizes to each pair of Label Extenders. Then, multiple Amplifier molecules hybridize to each Pre-Amplifier. Finally, multiple biotinylated Label Probe oligonucleotides hybridize to each Amplifier.
Step 4. Detection: Addition of streptavidin phycoerythrin (SAPE) generates a signal that is proportional with the amount of target RNA present in the sample. The signal is read using a Luminex instrument.
Step 5. Data Analysis: Analyze data using the Thermo Fisher Connect cloud-based QuantiGene data analysis application from any computer, anywhere. For advanced analysis and visualization, data can be analyzed using the free desktop application software, Applied Biosystems Transcriptome Analysis Console (TAC) software. TAC provides visualization and interpretation using tools such as scatter and volcano plots, hierarchical clustering, and provides link outs to publicly available annotations.
The QuantiGene Plex 384-well assay format enables multiplex gene expression analysis in a high throughput format. The assay uses target-specific probes to capture the RNA of interest and branched DNA technology to amplify fluorescent signals which are read with the FLEXMAP 3D, Luminex platform. Automation and batch processing options for this assay format increase sample throughput and decrease hands-on time while allowing for fast time to results for screening projects.
The QuantiGene Plex 384-well Assay Kit (Cat. No. QP1016) contains all the necessary reagents, buffers, and plates to perform QuantiGene Plex assays in 384-well plates. Design your target-specific QuantiGene Plex panel with our Panel Configuration Tool or contact your local Technical Sales Specialist for more information.
The QuantiGene Plex 384-well assay is designed to increase sample throughput and decrease hands-on time while allowing for fast time to results for screening projects. Below is example data that was generated using the 384-well format using the recommended equipment listed below.
Stimulation of cytokine production in HeLa cells
Cytokine production in cultured HeLa cells was stimulated using a Cell Stimulation Cocktail and monitored for various time points up to 24 hours. In this experiment, a full 80-plex panel was used for screening purposes focused on cytokine, and chemokine biomarkers.
Figure 1. Normalized gene expression of various targets in HeLa cells at different time points post stimulation with a Cell Stimulation cocktail. Y-axis: Average normalized expression; x-axis: mRNA targets; z-axis: time points of stimulation. Only a subset of gene targets tested is being displayed. HeLa cells were cultured in a 384-well format and stimulated with a Cell Stimulation cocktail for various times up to 24h (1h, 2h, 4h, 24h). Unstimulated cells served as baseline control. At the indicated time points, cells were lysed according to the QuantiGene sample prep protocol for cell lysates using liquid handling (Thermo Fisher Platemate). After all samples from all time points were prepared, the QuantiGene Plex 384-well assay was performed. Chart illustrates the normalized gene expression results.
The normalized gene expression of various targets in HeLa cells at different time points post stimulation with a Cell Stimulation cocktail reveals the degree of up- or downregulation. The average normalized expression (y-axis) represents the level of gene induction for the mRNA targets listed on the x-axis, while the time points of stimulation are shown on the z-axis. Of note, for example IL1B, IL8 and TGFB1 show pronounced relative gene expression at various time points of stimulation. While TGFB1 relative expression seems to gradually increase over time, peak expression for IL1B is seen at later time points around 24h. Table 1 shows normalized gene expression and fold change of various targets in HeLa cells at different time points post stimulation with a Cell Stimulation cocktail.
Table 1 and 2. Normalized gene expression and fold change of various targets in HeLa cells at different time points post stimulation with a Cell Stimulation cocktail.
Find instrumentation and equipment to make every step of your QuantiGene Plex 384-well workflow easy. For detailed options and recommendations for batch processing, refer to the QuantiGene Plex 384-well manual or contact your local Technical Sales Specialist.
Workflow step | Recommended equipment | Description | |
---|---|---|---|
Sample incubation and target hybridization | Temperature regulated orbital shaker: MaxQ 4450 Benchtop Orbital Shakers | ![]() | Versatile shaker ideal for incubating a small number of vessels and cell culture and many other applications. |
Plate washes | Magnetic microplate washers | Automated magnetic microplate washer for 384-well plates. | |
Sample incubation | Plate sealer: ALPS 50 V-Manual Heat Sealer | ![]() | Applies consistent, secure, tight seals around individual wells, eliminating sample loss through evaporation and cross contamination between wells. |
Sample and plate additions | Versette Automated Liquid Handler | ![]() | Compact liquid handler featuring user-friendly programming, reliable performance, and a choice of 96- or 384-channel pipetting heads. |
Multiplex detection | Luminex FLEXMAP 3D Instrument System | ![]() | Most advanced and multifunctional multiplex unit for simultaneous measurement of up to 80 tests in a single reaction volume. |
Parameter | 96-well | 384-well | |
---|---|---|---|
Product specification | Sensitivity (Limit of detection) | 1,000–2,000 transcripts/assay well | 2,000–4,000 transcripts/assay well |
Assay variation (C.V.) | ≤15% | ≤20% | |
Linearity | ≥3 logarithmic units | ||
Plex level | 3–80 | 3–80 | |
Samples | Cultured cells, bacteria, whole blood, PAXgene blood or dried blood spots, fresh/frozen tissues (animal or plant), FFPE samples, purified RNA | ||
Compatible Luminex instrumentation | MAGPIX, Luminex 200, FLEXMAP 3D, INTELLIFLEX | FLEXMAP 3D, INTELLIFLEX | |
Assay volumes | Target probe hybridization | 100 µL | 70 µL |
Pre-amp hybridization | 100 µL | 30 µL | |
Amp hybridization | 100 µL | 30 µL | |
Label probe hybridization | 100 µL | 30 µL | |
SAPE | 100 µL | 30 µL |
User manuals for use with assay kits QP1013, QP1014, QP1015 and QP1016
QuantiGene sample processing
Accelerate your research by outsourcing your gene expression profiling projects to our in-house Madison, WI scientists. Our assay service is designed to help you meet your specific project goals and timelines so you can free up your valuable resources for other discovery research.
Assay services features and benefits:
Design support—save time by accessing our bioinformatics team to customize your probe and panel design; obtain high-quality data with a fast turnaround time
Consistency—use the same customized assays in your follow-up studies that our team uses in the service
Flexibility—sample types include purified RNA, cultured cells, or tissue (whole blood, fresh or frozen tissue, or FFPE) pre-lysed in Invitrogen QuantiGene Buffer
Choice—a comprehensive list of target areas, including immunology, inflammation, cancer signalling, toxicology, neurology, cardiology, bone biology, metabolism, endocrinology, stem cell biology, apoptosis, and autophagy
Learn how to design a custom Invitrogen QuantiGene Plex Assay Panel for your research needs. This application note details recommendations for housekeeping genes and how to get started.
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For Research Use Only. Not for use in diagnostic procedures.