How does your assay work?

Our assay uses porous hydrogels made with our patented Optical Liquid Stamping methodology. Each particle has a specific fluorescent barcode mapped to a specific microRNA sequence. MicroRNAs hybridize to the probes throughout the volume of the detection region of the particle. Up to 70 different particle types can be mixed *in a single well* to allow high levels of multiplexing (68 probes of your choice and two controls). For more information see Our Technology.

Does the Firefly microRNA Assay distinguish different isoforms of the same microRNA?

The Firefly microRNA Assay detects target isoforms of + or - one base with the same probe at about 50% efficiency. For differences of more than one base, a new probe is required. This is ideal because, in general, researchers wish to pool all similar isoforms and look for how they change together. However, if you would like to distinguish similar isoforms, we can design custom probes to allow for that. Just contact technical support and request further information.

How does the Firefly microRNA detection limit compare to my RNA-seq data?

Because of the technical differences in the assays, there is not a perfect correlation between read numbers from RNA-seq data and detection limits for the Firefly MicroRNA Assay. For microRNAs with over 2000 reads, we routinely have very robust signal. We can often detect targets with as few as 50 reads. However, there is considerable variability from target to target for those sequences with less than 2000 reads.

What’s the best way to purify microRNAs for use with the Firefly MicroRNA Assay?

We highly recommend the Qiagen miRNeasy product line of kits for cells, tissue, serum, plasma, and FFPE tissues.Trizol extraction also works well.

The Norgen BioTek kit works but gives 3-5 fold lower signal than Qiagen and is therefore not recommended for low RNA content samples. Qiagen RNeasy Total RNA extraction kits preferentially select for RNAs longer than 200bp and have not been validated with our assay.

IMPORTANT NOTE: Life Technologies/Ambion miRVana kit is incompatible with our particles and cannot be used.

What controls should be included with the Firefly MicroRNA Assay?

Firefly includes in each well a positive control called “X-control”, to ensure that the reactions occurred robustly and consistently in each well, and a negative control “Blank” to control for background fluorescence. In addition, we recommend that you choose endogenous controls, microRNAs known not to fluctuate in your system, to allow for normalization between sample types and treatments. Firefly suggests the following endogenous controls for most samples but you should confirm that these work for your samples:

  • Human: RNU44, RNU48, RNU6B
  • Mouse: snoRNA202, snoRNA234
  • Cell Lines: miR-16-5p (same sequence for human, mouse, and rat) 
  • C. elegans: U18

For more detailed explanations see Controls.

Note: Serum and plasma do not contain RNUs so we recommend  miR-16, miR-451a, and mir-486 instead.

What’s the best way to choose the right microRNA targets?

Choosing the right targets for microRNA research used to require spending hours scanning the literature for all the right papers for your topic. We created a new, web-based tool - the Firefly Discovery Engine- to scan, parse, and summarize the literature for you. The Firefly™ Discovery Engine assembles a list of the most important microRNAs and associated genes from the scientific literature for any keyword or topic, putting years of research at your fingertips in seconds. The results can be downloaded as a .csv file for easy ordering or for further review.

What flow cytometers are compatible with the Firefly MicroRNA Assay?

Firefly particles are designed to be read on cytometers that have a blue (488nm) laser with green, yellow, and red detectors. Our microRNA assay has been optimized to work on the Millipore Guava easyCyte 8HT and Millipore Guava easyCyte 6HT, and the BD Accuri C6 benchtop cytometers. For details on calibrating these instruments for our assay click here.  Our assays are also supported for use on the Life Technologies Attune. If you’d like to find out about using our kits on a different cytometer please contact us. We also have an assay that works with the Millipore Muse. It uses the same types of particles and detection method as our standard particles except they have been optimized for the single laser format of the Muse. However, because the Muse has only a single laser, it can read only up to 36 total targets allowing for maximum multiplexing of 34 targets plus the two required internal controls.