Firefly particles | miRNA

FirePlex® particle technology

​​Find out how FirePlex 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 FirePlex particle technology that provides this multiplexing ability and allows assay readout on a standard flow cytometer.


FirePlex particles

​Particles are made from a porous bio-inert hydrogel that allows target capture throughout the 3D volume. Compared with the leading bead-based platform, FirePlex 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

  • Contain differing fluorescence intensities to create a fluorescent barcode
  • In a given panel, all particles that capture a particular miRNA contain the same code



​Central analyte quantification region

  • Contains probes to capture a specific target miRNA

Particle structure. Distinct regions capture target miRNAs and encode particles.


How are particles read by the flow cytometer?

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 FirePlex Analysis Workbench use information from the three regions to generate abundance data for each target miRNA:

  • Particle presence is triggered in the green channel. Events from all three regions are detected in the green channel to signify the presence of a particle.
  • Fluorescence intensity in the central region determines miRNA quantity. The flow cytometer detects a fluorescent signal in the red channel from the center of the of the particle. This comes from captured and labeled target miRNA and is proportional to the amount of miRNA present in the sample.
  • A fluorescent code to distinguish different miRNAs. By containing two end regions of varying fluorescence intensity, a particle code is generated in the yellow channel. Up to 70 codes are possible, and in every panel each miRNA is randomly assigned one of those codes so that data from a mixture of particles can be attributed to individual target miRNAs.
  • Software to stitch all the data together. After acquisition, the FirePlex Analysis Workbench software takes events from the three regions of the particles and stitches them back together into a single event, giving you simply the abundance data for each target miRNA.
Particle presence is triggered in the green channel. Events from all three regions are detected in the green channel to signify the presence of a particle.

Fluorescence intensity in the central region determines miRNA quantity. The flow cytometer detects a fluorescent signal in the red channel from the center of the particle. This comes from labeled target miRNA captured by the particle, and is proportional to the amount of miRNA present in the sample.

A fluorescent code to distinguish different miRNAs. By containing two end regions of varying fluorescence intensity, a particle code is generated in the yellow channel. Up to 70 codes are possible, and in every panel each miRNA is randomly assigned one of those codes so that data from a mixture of particles can be attributed to individual target miRNAs.

Software to stitch all the data together. After acquisition, the Firefly Analysis Workbench software takes events from the three regions of the particles and stitches them back together into a single event, giving you simply the abundance data for each target miRNA.


True multiplexing ability

FirePlex 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 FirePlex 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.