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Find out how Firefly particle technology works with your flow cytometer to allow highly sensitive, true multiplexed analysis.
Our multiplex miRNA assays allow simultaneous measurement of up to 68 target miRNAs in a single well, across many individual samples. At the heart of these assays is innovative Firefly particle technology that provides this multiplexing ability and allows assay readout on a standard flow cytometer.
Particles are made from a porous bio-inert hydrogel that allows target capture throughout the 3D volume. Compared with the leading bead-based platform, Firefly particle technology provides >100x more binding sites per particle. For nucleic acid capture and detection, this provides an additional two logs of dynamic range and ~75x increase in sensitivity.
Particles contain three distinct functional regions each separated from the other by inert spacer regions. The central analyte quantification region contains probes that capture target miRNAs. The two end regions function as two halves of a barcode to distinguish different particles.
End regions encode particles
Central analyte quantification region
Particle structure. Distinct regions capture target miRNAs and encode particles.
As particles pass through a flow cytometer they appear as a series of three cells. By separating the target quantification region from the particle encoding information, we reduce cross talk between channels and eliminate the need for compensation.
The flow cytometer and Firefly Analysis Workbench use information from the three regions to generate abundance data for each target miRNA:
Firefly particle technology allows true multiplexing; the ability to measure multiple miRNAs in the same well, without physical splitting of the sample. The particles enable this by containing the particle code to identify individual miRNA species and a separate analyte quantification region.
True multiplexing. Multiple miRNAs are detected within a single well.
A mixture of particles—each specific to a particular target miRNA—is added to each well to capture up to 68 target miRNAs. The Firefly Analysis Workbench software then differentiates between miRNAs using the particle code and combines this information with the signal from the central analyte quantification region to provide quantification data for individual miRNAs.
The particle mix added to each well contains on average 20 particles specific for each miRNA. This gives you 20 reads for every miRNA you measure and a maximum of 1,360 data points per well.