Graphical illustration of QuantiGene Plex technology that uses Luminex xMAP beads and branched DNA-based signal amplification to quantitate gene expression
Accurate and precise RNA quantitation—measure up to 80 genes in a single well

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.

Abgene™ 384 Well Polypropylene Storage Plates

New—QuantiGene Plex is now available in 384-well format for higher throughput. Get up to 30,720 data points per plate!

Learn more

Unique benefits of QuantiGene Plex Gene Expression Assay

  • Works with difficult sample types—works with degraded and cross-linked RNA in FFPE tissues and directly with blood; no RNA purification required
  • True multiplexing—measure up to 80 genes of interest and housekeeping genes in the same well with no cross-reactivity, reducing the number of samples needed
  • Standardized platform—96-well plate format compatible with Luminex 200, MAGPIX, and FlexMap3D systems and 384-well with FlexMap3D system
  • Simple workflow—ELISA-like workflow for target hybridization and signal amplification with no reverse transcription needed
  • Large inventory of validated genes—select from over 22,000 genes to create pathway- and disease-themed panels
  • Customization—if we don’t have your gene(s), we can create your custom panel within 3 weeks
     

How QuantiGene Plex Assay works

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. 

Video: QuantiGene Plex–How It Works animation

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.

QuantiGene Plex Gene Expression Assay workflow

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.

QuantiGene Plex Assay Workflow

Sample preparation for QuantiGene Plex assays is lysing samples to release stabilized RNAs. No RNA amplification is needed

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).

Target hybridization step using QuantiGene Plex assay involves incubating Luminex beads with capture probes with the target sample RNA and labeled extenders, blocking probes, and capture extenders
Signal amplification step in QuantiGene Plex assay involves adding the preamplifier, amplifier, and labeled probe to Luminex beads with capture probes containing target RNA

Step 3. Signal amplification: Signal amplification is achieved using branched DNA (bDNA) technology. First, an individual Pre-Amplifier molecule hybridizes to a 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.

Luminex 200 Instrument

QuantiGene Plex high throughput assays

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 faster 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.
 

QuantiGene Plex 384-well example data

The QuantiGene Plex 384-well assay is designed to increase sample throughput and decrease hands-on time while allowing for faster 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. 

See how scientists are using QuantiGene Plex 384-well in their research:

QuantiGene Plex instrumentation and equipment for high throughput and batch processing

Find instrumentation and equipment to make every step of your QuantiGene Plex 384-well workflow easier. 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 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 Thermo Scientific 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 Thermo Scientific Versette Automated Liquid Handler platform 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 Luminex FLEXMAP 3D Instrument System Most advanced and multifunctional multiplex unit for simultaneous measurement of up to 80 tests in a single reaction volume.

QuantiGene Plex specifications

  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 FLEXMAP 3D
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

QuantiGene Plex FAQs

  1. What sample types are compatible with QuantiGene Plex assay?
    Whole blood, fresh tissue, cultured cells, and purified RNA are validated. Other sample types including Tempus blood, bacteria, H&E stained/unstained FFPE, and blood spots should also be applicable.
  2. What data analysis software solutions are offered for the QuantiGene Plex assay?
    Analyze data using the QuantiGene cloud-based application from any computer, anywhere. For advanced analysis and visualize, data can be analyzed using the free desktop application software, Applied Biosystems Transcriptome Analysis Console (TAC) software.
  3. Can the VorTemp™ 56 Shaking Incubator be used for hybridization?
    We highly recommend the MaxQ Shaking incubator for optimal assay performance. It is possible to use the Vortemp, but 800 rpm is recommended.
  4. What is the recommended temperature for hybridization?
    The Day 1 temperature is 54°C while the Day 2 temperature is 50°C. Please use Quantigene Incubator Temperature Validation Kit (QS0517) to verify the incubator temperature.
  5. What are the proper storage conditions for the QuantiGene Plex reagents?
    When the kit arrives, please follow the instructions to store individual components. The Preamp/Amp/LP Diluent, Bead Mix, SAPE, SAPE Diluent should be stored at 4°C, while probe set mix, Proteinase K, and blocking reagent should be stored at -20°C. Others are stored at room temperature. Refer to the product insert for details.
  6. What reagents are included in the kit?
    The kit provides all necessary reagents to perform the assay except Target Probe and Bead mix which are ordered separately. See the user manual for a list of the reagents.
  7. Can QuantiGene Plex 384-well assays be used for miRNA or DNA analysis?
    No. We have options with the QuantiGene Singleplex assay for miRNA and DNA.
  8. Should any control/housekeeping genes be used in my panel
    Three or more control/housekeeping genes are recommended to normalize the gene expression results.
  9. Can I use a heat sealer to seal the plate on day 2 of the assay
    No, manual sealing using the provided seal is recommended. Heat sealing would deform the plate causing problem in washing.
  10. How do I dilute my samples?
    For cell lysate or blood lysate, please use Diluted Lysis Mix (DLM); for tissue homogenates, please use Homogenizing Solution.
  11. How should I seal the Day 1 plate for Target Probe hybridization?
    The Day 1 plate can be sealed either manually or using a heat sealer. The pressure seal for manual sealing and the heat seal AB-0757 for Thermo Fisher ALPS 50 V (AB-1443A) are provided in the kit. If a different heat sealer will be used, please consult the manufacturer for suitable sealing.

Unique benefits of QuantiGene Plex Gene Expression Assay

  • Works with difficult sample types—works with degraded and cross-linked RNA in FFPE tissues and directly with blood; no RNA purification required
  • True multiplexing—measure up to 80 genes of interest and housekeeping genes in the same well with no cross-reactivity, reducing the number of samples needed
  • Standardized platform—96-well plate format compatible with Luminex 200, MAGPIX, and FlexMap3D systems and 384-well with FlexMap3D system
  • Simple workflow—ELISA-like workflow for target hybridization and signal amplification with no reverse transcription needed
  • Large inventory of validated genes—select from over 22,000 genes to create pathway- and disease-themed panels
  • Customization—if we don’t have your gene(s), we can create your custom panel within 3 weeks
     

How QuantiGene Plex Assay works

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. 

Video: QuantiGene Plex–How It Works animation

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.

QuantiGene Plex Gene Expression Assay workflow

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.

QuantiGene Plex Assay Workflow

Sample preparation for QuantiGene Plex assays is lysing samples to release stabilized RNAs. No RNA amplification is needed

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).

Target hybridization step using QuantiGene Plex assay involves incubating Luminex beads with capture probes with the target sample RNA and labeled extenders, blocking probes, and capture extenders
Signal amplification step in QuantiGene Plex assay involves adding the preamplifier, amplifier, and labeled probe to Luminex beads with capture probes containing target RNA

Step 3. Signal amplification: Signal amplification is achieved using branched DNA (bDNA) technology. First, an individual Pre-Amplifier molecule hybridizes to a 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.

Luminex 200 Instrument

QuantiGene Plex high throughput assays

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 faster 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.
 

QuantiGene Plex 384-well example data

The QuantiGene Plex 384-well assay is designed to increase sample throughput and decrease hands-on time while allowing for faster 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. 

See how scientists are using QuantiGene Plex 384-well in their research:

QuantiGene Plex instrumentation and equipment for high throughput and batch processing

Find instrumentation and equipment to make every step of your QuantiGene Plex 384-well workflow easier. 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 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 Thermo Scientific 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 Thermo Scientific Versette Automated Liquid Handler platform 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 Luminex FLEXMAP 3D Instrument System Most advanced and multifunctional multiplex unit for simultaneous measurement of up to 80 tests in a single reaction volume.

QuantiGene Plex specifications

  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 FLEXMAP 3D
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

QuantiGene Plex FAQs

  1. What sample types are compatible with QuantiGene Plex assay?
    Whole blood, fresh tissue, cultured cells, and purified RNA are validated. Other sample types including Tempus blood, bacteria, H&E stained/unstained FFPE, and blood spots should also be applicable.
  2. What data analysis software solutions are offered for the QuantiGene Plex assay?
    Analyze data using the QuantiGene cloud-based application from any computer, anywhere. For advanced analysis and visualize, data can be analyzed using the free desktop application software, Applied Biosystems Transcriptome Analysis Console (TAC) software.
  3. Can the VorTemp™ 56 Shaking Incubator be used for hybridization?
    We highly recommend the MaxQ Shaking incubator for optimal assay performance. It is possible to use the Vortemp, but 800 rpm is recommended.
  4. What is the recommended temperature for hybridization?
    The Day 1 temperature is 54°C while the Day 2 temperature is 50°C. Please use Quantigene Incubator Temperature Validation Kit (QS0517) to verify the incubator temperature.
  5. What are the proper storage conditions for the QuantiGene Plex reagents?
    When the kit arrives, please follow the instructions to store individual components. The Preamp/Amp/LP Diluent, Bead Mix, SAPE, SAPE Diluent should be stored at 4°C, while probe set mix, Proteinase K, and blocking reagent should be stored at -20°C. Others are stored at room temperature. Refer to the product insert for details.
  6. What reagents are included in the kit?
    The kit provides all necessary reagents to perform the assay except Target Probe and Bead mix which are ordered separately. See the user manual for a list of the reagents.
  7. Can QuantiGene Plex 384-well assays be used for miRNA or DNA analysis?
    No. We have options with the QuantiGene Singleplex assay for miRNA and DNA.
  8. Should any control/housekeeping genes be used in my panel
    Three or more control/housekeeping genes are recommended to normalize the gene expression results.
  9. Can I use a heat sealer to seal the plate on day 2 of the assay
    No, manual sealing using the provided seal is recommended. Heat sealing would deform the plate causing problem in washing.
  10. How do I dilute my samples?
    For cell lysate or blood lysate, please use Diluted Lysis Mix (DLM); for tissue homogenates, please use Homogenizing Solution.
  11. How should I seal the Day 1 plate for Target Probe hybridization?
    The Day 1 plate can be sealed either manually or using a heat sealer. The pressure seal for manual sealing and the heat seal AB-0757 for Thermo Fisher ALPS 50 V (AB-1443A) are provided in the kit. If a different heat sealer will be used, please consult the manufacturer for suitable sealing.

QuantiGene Plex Assay services

Let us test and analyze your samples to save you time 

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

Request a quote


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