Detect RNA and protein simultaneously by flow cytometry

Flow cytometry, with its ability to acquire and analyze millions of individual single cells simultaneously, to use multiplexing capabilities and to detect both cell surface and intracellular proteins in a straightforward workflow, has long been the standard for characterizing heterogeneous cell populations. Nevertheless, flow cytometry has been constrained by the availability and adequacy of antibodies to measure all analytes of interest. MicroRNA and long non-coding RNA, messenger RNA, viral transcripts, unique model organisms, and targets for which antibody development is troublesome have not been able to utilize the power of flow cytometry, and have historically required that researchers conduct numerous disconnected experiments to analyze these cellular systems.

With Invitrogen PrimeFlow RNA Assay, researchers can now reveal the dynamics of RNA and protein expression simultaneously within millions of single cells. This novel assay employs fluorescent in situ hybridization (FISH) with the power of branched DNA (bDNA) amplification technique for simultaneous detection of up to four RNA targets in combination with immunophenotyping for cell surface and intracellular proteins using fluorochrome-conjugated antibodies. Data are collected using a standard flow cytometer.

The PrimeFlow RNA Assay workflow.


Technology overview

In the PrimeFlow RNA Assay, branched-DNA technology is used to amplify the detection of a RNA transcript, rather than the target RNA itself. The PrimeFlow RNA Assay can detect mRNA, long non-coding RNA, and microRNA, as well as viral RNA and telomere DNA. Detection of microRNA is optimal when using the Invitrogen PrimeFlow microRNA Pretreatment Buffer (Cat. No. 88-18006) and protocol. In the first hybridization step of the assay, a gene-specific oligonucleotide Target Probe set that contains 20–40 probe pairs (or a single pair in the case of microRNA probe sets) binds to the target RNA sequence. An individual probe pair is designed to bind adjacent to each other in order for signal amplification to take place. Signal amplification is then achieved through a series of sequential hybridization steps. The PreAmplifier molecules confer an additional level of specificity because they will hybridize to the Target Probes only when both halves of a respective probe pair have bound to their target sequence. Multiple Amplifier molecules subsequently hybridize to their respective PreAmplifier.

Finally, Label Probe oligonucleotides conjugated to a fluorescent dye hybridize to their corresponding Amplifier molecules. A fully assembled signal amplification "tree" has 400 Label Probe binding sites. When all target-specific oligonucleotide probes in the Target Probe set bind to the target RNA transcript, 8,000–16,000-fold amplification is achieved.

We currently offer four unique amplification structures that allow simultaneous measurement of up to four different RNA targets for multicolor flow cytometric analysis. Four types of probe sets are currently available to allow detection of RNA labeled with Alexa Fluor 647 (Type 1 Probe Sets), Alexa Fluor 488 (Type 4 Probe sets), or Alexa Fluor 568 (Type 10 Probe Sets). When detecting more than one RNA target in a single sample, each Probe Set must be a unique type to differentiate its signal from the others. Once the cells have been processed by the PrimeFlow RNA Assay, the data can be collected and analyzed on any standard flow cytometer. The schematic above illustrates the detection of two unique targets.

Search for available (in stock) probe sets

Assay kits and supporting products

This PrimeFlow RNA Assay contains all the reagents needed to conduct the assay. Positive control probes and target probes for genes of interest are sold separately.

Key publications

  1. Baxter AE, Niessl J, Fromentin R et al (2016) Single-cell characterization of viral translation-competent reservoirs in HIV-infected individuals. Cell Host Microbe 20(3):368–380.

  2. Soh KT, Tario JD Jr, Colligan S et al (2016) Simultaneous, single-cell measurement of messenger RNA, cell surface proteins, and intracellular proteins. Curr Protoc Cytom 75:7.45.1–7.45.33.

  3. Shalapour S, Font-Burgada J, Di Caro G et al (2015) Immunosuppressive plasma cells impede T-cell-dependent immunogenic chemotherapy. Nature 521(7550):94–98.

  4. Wojno ED, Monticelli LA, Tran SV et al (2015) The prostaglandin D2 receptor CRTH2 regulates accumulation of group 2 innate lymphoid cells in the inflamed lung. Mucosal Immunol 8(6):1313–1323.

  5. Porichis F1, Hart MG2, Griesbeck M et al (2014) High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry. Nat Commun 5:5641.
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Elia Tait Wojno, Assistant Professor, Cornell University College of Veterinary Medicine
Detection of RNA with Flow Cytometry by Dr. Porichis