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
Principle behind the PrimeFlow RNA assay
- Gene specific oligonucleotide target probe set binds to the target RNA sequence
- Preamplifier and multiple amplifier molecules subsequently hybridize to target RNA for signal amplification
- Label probe oligonucleotides conjugated to fluorescent dye detect up to four RNA targets per cell by flow cytometry
Featured PrimeFlow Assay applications
Follow the five steps below to begin your PrimeFlow RNA Assay
|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|
- 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.
5 Steps Resources
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For Research Use Only. Not for use in diagnostic procedures.