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PrimeFlow® RNA Assay Principle

PrimeFlow RNA Assay video

Overview: PrimeFlow® RNA Assay technology

Fluorescent in situ hybridization (FISH) is a powerful technique that allows specific localization of ribonucleic acid targets in fixed cells. The basic premise of the application relies on detecting nucleic acids through sequential hybridization of nucleic acid probes, that provides to obtain gene expression information at a single cell level. Current FISH techniques are generally limited by high background and low sensitivity due to non-specific binding and inefficient signal amplification.

PrimeFlow® RNA Assay incorporates a proprietary oligonucleotide probe set design and branched DNA (bDNA) signal amplification technology to analyze RNA transcripts by flow cytometry. bDNA technology provides a unique approach to RNA detection and signal amplification by amplifying the reporter signal rather than the target sequence (e.g. PCR) for consistent results, a common problem for PCR-based assays.

In the PrimeFlow® RNA Assay, target-specific probe sets contain 20-40 oligonucleotide pairs that hybridize to the target RNA transcript. Signal amplification is achieved through specific hybridization of adjacent oligonucleotide pairs to bDNA structures, formed by pre-amplifiers, amplifiers and fluorochrome-conjugated label probes, resulting in excellent specificity, low background and high signal-to-noise ratio.

PrimeFlow® RNA Assay Workflow

PrimeFlow® RNA Assay principle

The assay workflow contains several steps: surface antibody staining, fixation and permeabilization, intracellular antibody staining, followed by target probe hybridization with RNA-specific probe sets, signal amplification using bDNA constructs, and detection by flow cytometry. For simplicity, detection of only two RNA targets are shown in orange and yellow (in the figure above) with only three of the 20-40 oligonucleotide target probe pairs per target RNA.

Sample preparation: Antibody staining, fixation and permeabilization

Single-cell suspensions can be stained for cell surface markers and fixable viability dyes, as before the cells are fixed and permeabilized. Subsequently, the cells may be stained with antibody directed to intracellular targets, such as transcription factors and cytokines. After an additional fixation step, the cells are ready to proceed through the hybridization and signal amplification steps.

Target hybridization

A target-specific Probe Set contains 20-40 oligonucleotide pairs that hybridize to specific regions across the target RNA sequence. Subsequent signal amplification requires that each half of a given oligonucleotide pair binds to the target RNA in adjacent positions. Three 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® 750 (Type 6 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.

Probe Set Type

Fluorochrome Label

Excitation Wavelength(max)

Emission Wavelength (max)

Laser Excitation Wavelength

Bandpass Filter Recommendation

Type 1

Alexa Fluor® 647

647 nm

668 nm

632-640 nm

660/20

Type 4

Alexa Fluor® 488

488 nm

519 nm

488 nm

530/30

Type 6

Alexa Fluor® 750

749 nm

775 nm

632-640 nm

780/60

Signal amplification

Signal amplification using bDNA technology is achieved through a series of sequential hybridization steps, that forms a "tree" like structure. Pre-amplifier molecules hybridize to their respective pair of bound oligonucleotide probes to form the “trunk” of the tree. Multiple amplifier molecules hybridize to their respective pre-amplifier to create the “branches.” Finally, multiple Label Probes hybridize to the Amplifiers and form the "leaves" of the "tree." A fully assembled signal amplification tree contains 400 label probe binding sites. If all target-specific oligonucleotides in the probe set bind to the target RNA transcript, an 8,000 fold amplification can be achieved.

Fluorescence detection

Upon completion of the assay protocol, target RNA data is detected in cells by analyzing the sample on a standard flow cytometer equipped with a 488 nm laser and appropriate filter configurations, to capture the fluorescent signals.