Firefly microRNA Assay



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Firefly MicroRNA Assay Performance


The Firefly™ microRNA Assay offers high sensitivity and broad dynamic range
-- The Firefly™ microRNA Assay is a versatile, customizable microRNA assay with sub-attomole sensitivity (a detection limit of ~200,000 molecules) and broad dynamic range that can be used directly in cell lysate and tissue digest.  The assay has been validated against qPCR, microarray, and northern blot data and is the next generation in microRNA detection. Unlike many other technologies, the assay is rapid, high-throughput, and requires minimal hands-on time with few pipetting operations.

The hydrogel particles used in the Firefly™ microRNA Assay enable efficient capture of your target molecules throughout the entire particle volume, not just on the surface like competing assays.  This means you can expect consistent expression results over a broad range of assay input amount, as shown on the right. 

 

 


The Firefly™ microRNA Assay has single-nucleotide specificity--The Firefly™ microRNA Assay can be used for any organism and any species because of its high specificity.  microRNA expression results from a total RNA extraction of C.elegans (left) show no crosstalk between microRNAs that vary by only 1-2 nucleotides.

The Firefly™ microRNA Assay detects microRNA directly in a variety of biological samples--With the Firefly™ Assay, you can expect microRNA expression results to be representative of your original sample--tumor biopsies, tissue samples, cell culture--regardless of whether you perform an RNA extraction.  

             

Compatibility with Standard Benchtop Cytometers--the Firefly™ microRNA Assay readily expands the capabilities of almost any cytometer. We support many instruments from BD, Millipore, and Miltenyi. You can read more about support for specific instruments and our expanding capabilities here.

  

 

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

Trizol extraction also works well. For Trizol purifications, glycogen does not interfere with the assay. We recommend precipitating with ethanol.

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.

DESIGNING RELIABLE EXPERIMENTS
To obtain reliable microRNA expression data, it is important that an experiment be designed to accurately subtract background signal, allow for appropriate normalization, and assess inter-well variability. Ideally, every experiment performed with the Firefly™ microRNA Assay includes (1) positive and blank controls in each well, (2) negative control wells, and (3) biological or technical replicates.

(1) Controls within each well
Firefly BioWorks utilizes both a positive control (“X-Control”) and a blank (“Blank”) by default in each custom panel. In addition to these, it is strongly recommended that users include at least one endogenous control.

Positive control particles bear probes for a miRNA-like target, X-control, that is present in Firefly Hybe Buffer at a concentration of ~1 fmol per 25 μl. This control gives confidence that the assay was successfully implemented in every well.

BLANK particles bear no probe, giving a baseline level of the background fluorescence in every assay well. This signal should be subtracted from those obtained by other targets in the same well.

Endogenous controls are small nucleolar RNAs or microRNAs expressed at consistent levels under a variety of conditions across tissues. These controls are important for signal normalization between sample types and treatments. Users are given the freedom to select endogenous controls most appropriate for their applications and may prefer to include more than one endogenous control in their panel. Firefly suggests the following endogenous controls:
           Human: RNU44, RNU48, RNU6B
           Mouse: snoRNA202, snoRNA234 
           Cell Lines: miR-16-5p (same sequence for human, mouse, and rat)
           C. elegans: U18

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

(2) Negative control wells


In order to obtain an accurate measurement of the background signal for each microRNA in a panel, it is necessary to run negative control wells, where carrier buffer is used in place of a biological sample. Furthermore, the use of multiple negative control wells allows users to estimate inter-well variability, giving more confidence to the results obtained. Firefly BioWorks strongly recommends the use of at least three negative controls every time an assay is performed.  An example of negative control wells is provided in the sample plate layout below.

 

(3) Replicates
The use of replicates gives statistical meaning to results by, for example, enabling the calculation of mean and standard deviation. Replicates can be performed at the stage of sample preparation (biological) or assay (technical).

Biological replicates are those in which the same conditions are used to treat and prepare samples from different sources. These replicates are important in determining the biological variation within a population. An example of this type of replicate is serum samples derived from 3 different mice injected with the same TNF inhibitor.

Technical replicates are those in which samples derived from the same source are assayed multiple times. These replicates are important in determining reproducibility of the assay. Ideally, every assay has technical replicates.

For questions, please inquire!

Vacuum Manifold for 96-well Filter Plates



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Shaking Incubator

Supported Cytometers

(Firefly BioWorks does not distribute flow cytometers)

Firefly particles are designed to be read on cytometers that have a blue (488nm) laser with green, yellow, and red detectors. The benchtop cytometers that FirePlex® miRSelect has been optimized to work with include:

 

Millipore Muse

FirePlex® miRSelect calibration and usage guides for these cytometers are available here.

FirePlex® miRSelect has also demonstrated multiplexed detection using larger core facility machines. Our products are approved for use with the following machines:

Life Technologies Attune

For other machine configurations, please contact us!

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.

Official protocols, tutorials, and product sheets available to study Firefly BioWorks' complete solution to multiplexed microRNA detection. 

Firefly™ Discovery microRNA Assay Guides and MSDS (formerly called FirePlex® miRSelect)      

       Visual Protocol         MSDS

       Firefly™ microRNA Assay Panel Design Guide 

       Firefly™ microRNA Assay Video Protocol


Firefly™ Analysis Workbench (data analysis software)

     Click here to download the Firefly™ Analysis Workbench.

       User Guide 

       Quick-Start Guide 

      Sample data download

 

Vacuum Manifold
     
   Product Sheet  


Cytometer Calibration and Operation Procedures    
     
 BD Accuri C6-High Throughput    

      BD Accuri C6-Manual Sample Analysis

      Life Technologies Attune      Attune Settings Files

      Millipore Guava 6HT/8HT  

      Guava 6HT Settings Files        Guava 8HT Settings Files        Guava 12HT Settings Files