QuantiGene® FlowRNA

QuantiGene® FlowRNA Technology

In Situ Hybridization (ISH) is a powerful technique that allows specific localization of nucleic acid targets in fixed cells.  The basic premise of the application relies on detecting nucleic acids when they are appropriately fixed and applying a nucleic acid probe, allowing one to obtain gene expression information at a single cell level.

Branched DNA (bDNA) technology is a nucleic acid hybridization assay that provides a unique approach for RNA detection and quantification by amplifying the reporter signal rather than the target sequence.

Branched DNA (bDNA) Probe Design Principle

QuantiGene FlowRNA bDNA

The QuantiGene FlowRNA Assay is an ISH technique incorporating a proprietary oligo probe set design and bDNA signal amplification technology to analyze RNA transcripts by flow cytometry.  A typical target-specific probe set containing 20 oligo pairs hybridizes to the target RNA. Signal amplification is detected through specific hybridization of an adjacent probe set of oligo pairs to a target RNA.  The result is RNA molecule-level data with excellent specificity, low background and high signal-to-noise ratio.

The QuantiGene FlowRNA Assay Workflow

QuantiGene FlowRNA Assay Principle

QuantiGene FlowRNA Assay Principle

The assay workflow contains four steps: sample preparation, target hybridization with probes, signal amplification using bDNA technology, and detection. For simplicity, two RNA targets are shown (orange and yellow) and only three of the 20 oligo pairs per target RNA.

Sample Preparation*

Cells in suspension are fixed, then permeabilized to allow target accessibility.

Target Hybridization

A target-specific probe set containing 20 oligo pairs is hybridized to the target RNA sequence. Subsequent signal amplification is predicated on specific hybridization of adjacent oligo pairs to a target RNA, or in simpler terms, in order for a signal amplification branch to bind to the target RNA, both oligos from the oligonucleotide pair are required to hybridize side by side to the target RNA.

Signal Amplification

Signal amplification using bDNA technology is achieved through a series of sequential hybridization steps.  PreAmplifier molecules hybridize to their respective pair of bound probe set oligos, then multiple Amplifier molecules hybridize to their respective PreAmplifier. Next, multiple Label Probe oligos, conjugated to fluorescent dye, hybridize to their corresponding Amplifier molecule.  A fully assembled signal amplification "tree" has 400 Label Probe binding sites. When all target-specific oligos in the Probe Set bind to the target mRNA transcript, an 8,000 fold amplification is achieved.  Currently, up to three compatible fluorochrome-labeled amplification “tree” structures allow simultaneous measurement of up to three different RNA targets.

Fluorescence Detection

Once cells have been processed through the assay protocol steps, target RNA data can be collected and analyzed on a flow cytometer with proper filter configurations to capture appropriate fluorescent signals.

*Antibody detection of surface markers or inclusion of viability dyes requires pre-labeling cells with antibody conjugated to organic dyes or amine-reactive viability dyes.  Examples of dyes and fluorochromes are listed in the FAQ section.

Specificity Validation

QuantiGene FlowRNA bDNAQuantiGene Image 4

QuantiGene Image 5

QuantiGene FlowRNA Specificity Validation

The bDNA technology achieves high target specificity with the use of oligonucleotide pairs – a design resulting in signal amplification only when two adjacent target probe oligos (left oligo and right oligo) bind to the specific target (top figure). To determine the assay specificity, probe sets for human GAPDH containing left oligo alone or right oligo alone are tested and compared to a complete probe set in human U937 cells (middle & bottom figures). Strong signal is detected with the complete probe set, correlating to the abundant expression of GAPDH in U937 cells. In contrast, no significant signal is detected when either left oligos or right oligos are used individually as compared to the “no probe” control. A negative control with a complete probe set (DapB gene in Gram-positive bacterium, Bacillus subtillis) yielded no specific signal.