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RNA Detection By Flow Cytometry
Detecting RNA Targets by Flow Cytometry
Invitrogen PrimeFlow RNA Assay expands the capability of flow cytometry to measure RNA. Combine PrimeFlow with immunolabeling of both cell-surface and intracellular proteins using fluorophore-conjugated antibodies. Researchers now can reveal the dynamics of RNA transcription together with protein expression patterns in single cell resolutions
Find PrimeFlow probes for your target–including SARS-CoV-2
Ready to start the PrimeFlow RNA Assay? Check what you’ll need
Principle behind the PrimeFlow RNA assay
In the PrimeFlow RNA Assay, target-specific probe sets hybridize to the target RNA transcript. Signal amplification is achieved through specific hybridization of adjacent oligonucleotide pairs to bDNA structures, formed by preamplifiers, amplifiers, and fluorescently labeled probes, resulting in excellent specificity, low background, and high signal-to-noise ratios.
Featured PrimeFlow Assay applications
Evaluate virus RNA in infected cells
Detect microRNA
Probe mRNA when no antibody exists to detect the protein
Not finding the application you're looking for? Let us know what you need
Ordering information
Follow the five steps below to begin your PrimeFlow RNA Assay
| Instrument | Specification |
|---|---|
| Flow cytometer | Lasers:
Filter sets:
|
| Incubator | Capable of maintaining temperature at 40 ±1°C |
| Metal heat block | For 1.5 mL microcentrifuge tube, placed inside the validated incubator |
| Temperature validation kit | The Invitrogen ViewRNA Temperature Validation Kit uses a calibrated thermometer to verify the incubator temperature |
Optional: Is your target microRNA? This buffer helps ensure you get improved signal and better sensitivity for miRNA.
Optional: Will the assay be processed in a 96-well plate?
- Four types of probe sets are currently available for RNA detection
- Select different types of probe set depending on the RNA expression level
- For multiplex analysis with immunolabeling of both cell-surface and intracellular proteins, find fluorophore-conjugated antibodies
| Expression level of target RNA | Fluorochrome label | Laser excitation wavelength (nm) | Bandpass filter (nm) | Fluorescence channel | Probe set type |
|---|---|---|---|---|---|
| Low | Alexa Fluor 647 | 633, 635 | 660/20 | APC, Alexa Fluor 647, eFluor 660 | Type 1 |
| Low | Alexa Fluor 568 | 561 | 610/20 | PE-eFluor 610, Alexa Fluor 568, PE-Texas Red | Type 10 |
| Medium-to-high | Alexa Fluor 488 | 488 | 530/30 | FITC, Alexa Fluor 488 | Type 4 |
| Medium-to-high | Alexa Fluor 750 | 633, 635 | 780/60 | APC-Cy7, APC-eFluor 780, Alexa Fluor 750 | Type 6 |
Search for available (in stock) probe sets
Not finding what you're looking for? Go to Custom Branched DNA Probe Set Tool
- Positive control—use positive control probe sets in every experiment to ensure proper assay performance: RPL13a for human leukocytes and β-actin (ACTB) for mouse tissues; for specific cell types and other recommended genes, refer to Appendix 5 in the product manual of the PrimeFlow RNA Assay Kit
- Negative control—such as samples with the target specific probe omitted, or samples labeled with a probe against a target not expressed in the cells of interest (e.g., dapB, a bacterial gene); highly recommended
Search probes used for positive and negative controls in probe search tool in Step 4.
Do you need SARS-CoV-2 probes?
Use the step-by-step ordering information above to order your kit and probes, including controls. In step 4, if you need SARS-CoV-2 probes, see the table below, and follow the ‘How to order SARS-CoV-2 probes’ instructions provided.
| Dye | Type | Scale | Cat. No. | Assay ID |
|---|---|---|---|---|
| AF647 | 1 | 40 assays | CPF-204 | VPNKRHH |
| AF647 | 1 | 100 assays | CPF-210 | VPNKRHH |
| AF488 | 4 | 40 assays | CPF-204 | VP9HHYZ |
| AF488 | 4 | 100 assays | CPF-210 | VP9HHYZ |
| AF750 | 6 | 40 assays | CPF-204 | VPAAADR |
| AF750 | 6 | 100 assays | CPF-210 | VPAAADR |
| AF568 | 10 | 40 assays | CPF-204 | VPCE3XN |
| AF568 | 10 | 100 assays | CPF-210 | VPCE3XN |
PrimeFlow probe sets were designed using the NCBI strain and sequence from Accession Number LC521925, and cover multiple regions of the viral transcript. Find in the table below the binding sites and transcript region for the probe sets included in the assay. If you need a custom probe designed for a specific RNA region, use the Custom Branched DNA Probe Set Tool.
| Position (5' to 3') | Region | |
|---|---|---|
| 1 | 705-1676 | orf1ab |
| 2 | 4356-5607 | orf1ab |
| 3 | 12808-13942 | orf1ab |
| 4 | 20571-21759 | orf1ab |
| 5 | 26646-27763 | M |
| 6 | 28212-29152 | ORF8 and N |
Webinars
PrimeFlow resources
Related products
Resources
Fluorophore and reagent selection guide for flow cytometry
Download Flow Cytometry Protocols Handbook
Spectral Flow Cytometry Fundamentals
Invitrogen eBioscience Resources—Selection guides, Best Protocols, product performance and more.
Intracellular Staining for Flow Cytometry How-To Video—for detecting cytokines and intranuclear markers.
Flow Cytometry Learning Center—Access flow cytometry educational resources for better experiment planning and execution.
Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs.
5 Steps Resources
Support
Flow Cytometry Support Center—Find technical support recommendations for your flow cytometry workflows, including tips for experimental setup and in-depth troubleshooting help.
Flow Cytometry Panel Design Support—Work with one of our technical sales specialists to discuss your experimental needs and guide you through the process.
Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.