Firefly particles

FirePlex® miRNA assay

The FirePlex® miRNA assay enables biomarker discovery and verification studies directly from small volumes of biofluid, without need for RNA purification or pre-amplification.  

Jump to:

1. Benchmarking the FirePlex miRNA assay
2. FirePlex miRNA assays FAQ
3. RNA purification sample preparation guidelines
4. Equipment for FirePlex miRNA assays
5. FirePlex miRNA assay controls
​6. How does the FirePlex miRNA work?
7. MicroRNA profiling methods: comparison of current technologies
8. MicroRNA profiling direct from biofluids, using FirePlex particle technology
9. FirePlex miRNA disease focus panels

FirePlex miRNA assay guide

>>Next page: Custom miRNA panels

A customizable biomarker development platform

The FirePlex miRNA assay is designed for both discovery and verification studies. Select between pre-designed panels for key research areas, larger discovery panels, or design custom panels to detect any miRNA from any species, whether annotated in miRBase or not. 

Direct from crude biofluid samples

Robust miRNA profiles can be obtained directly from crude sample types including serum, plasma, crude exosomes, FFPEs, cell suspensions, urine and saliva. Measurement directly from biofluids using the FirePlex miRNA assay prevents sample bias, simplifies assay workflow and reduces running time. 

Figure 1.  Assay sensitivity in crude biofluid and purified RNA. To demonstrate the high sensitivity of the FirePlex miRNA assay, we measured the number of miRNAs detected from varying input amounts of pooled human serum or pooled RNA from human brain, lung and liver samples. We robustly detect most targets from the 48-plex panel in as little as 10 µL of serum or 100 pg of RNA​.

Small sample input

Profile miRNAs even when sample is in short supply. Detect up to 65 miRNAs per well with PCR sensitivity from 10 µL biofluid or 100 pg RNA inputs.

Integrated software

To aid custom panel design, we provide an online tool – the FirePlex Discovery Engine. This allows you to perform species-specific literature searches to identify miRNAs implicated in diseases or research areas of interest. 

In addition, our integrated FirePlex Analysis Workbench software enables easy and rapid data analysis, visualization, and export in under ten minutes, and includes key features such as standard curve analysis and publication-quality heat maps and graphs.

Find out more


Benchmarking the FirePlex®​ miRNA assay 

FirePlex miRNA assays demonstrate equivalent or better performance compared to currently available miRNA profiling technologies.

The full potential of miRNA profiling is currently limited by assay platform performance standards. Current challenges include the limited throughput of existing validation technologies, errors introduced during sample purification and insufficient methods used for analytical normalization and quality control.

FirePlex particle technology provides new solutions to these limitations, finally enabling researchers to rapidly and cost-effectively evaluate miRNA biomarker signatures directly from biofluids, and in solid tissues. With the capability for high-performance, high-throughput multiplexing and readout on a standard flow cytometer, the true potential of miRNA profiling is now accessible to the scientific community.

In this technical note, we demonstrate the excellent reproducibility of FirePlex miRNA assays, even with diminishing RNA input, with equivalent or better performance compared to currently available miRNA profiling technologies.

Read this technical note in full.

You can also learn more about miRNA research, here.

Contact us at FirePlexSales@abcam.com


FirePlex® miRNA assays FAQ 

Our FAQ answers your most common questions for the FirePlex miRNA assay.

Contents

​General

  • 1) What is true multiplexing?
  • 2) I don’t know which microRNA to study. How can I choose the correct ones?
  • 3) How many miRNAs can be detected using this assay?
  • 4) I have small quantity of RNA isolated from cells in culture, which kit is the most suitable for me? ​
  • 5) Can I run the assay with just the core kit?
  • 6) What do I need to run the assay?
  • 7) How long does the assay take to run?
  • 8) What is the sensitivity of the assay?
  • 9) What is the reproducibility of the assay?
  • 10) How do I analyze the data?
  • 11) How does this technology differ from bead based assays?
  • 12) Can I normalize my data using a synthetic target spiked into the serum or plasma samples?​


​Controls

  • 13) Which controls are included in the assay?
  • 14) Should I include additional controls?

Sample

  • 15) What sample types are compatible with the assay? 
  • 16) I want to run sample type that is not listed as tested yet, can I do that?
  • 17) How much sample/starting material is required?
  • 18) How many samples can I run at once?
  • 19) How should I prepare the sample? 


1) What is true multiplexing?
We use the term ‘true multiplex’ to reflect that our miRNA assay can simultaneously measure up to 65 miRNAs in every well. This approach differs from parallel miRNA detection in which samples are split across many wells before detection of a single target per well. Learn more about the technology.

2) I don’t know which microRNA to study. How can I choose the correct ones?
The FirePlex Discovery Engine helps you find important miRNAs for any topic. Simply insert your keywords and select from a range of species, including human, mouse and fly. After scouring the scientific literature, the Discovery Engine generates a list of relevant miRNAs ranked according to number and impact of publications. To find more information, you will find links straight through to PubMed and miRBase from each miRNA entry.

Additionally, we have compiled a miRNA resources page, where you can find a selection of databases and target predictor tools.

We also offer convenient miRNA focus panels where we’ve chosen 65 targets from peer-reviewed studies that have been shown to be differentially regulated in different diseases.

3) How many miRNAs can be detected using this assay?
Up to 65 miRNAs per well can be detected with this assay.

4) I have a small quantity of RNA isolated from cells in culture, which kit is the most suitable for me?
If you have 100 pg–500 ng (in a 25 μL volume) we recommend use of the FirePlex miRNA Assay for purified RNA.

5) Can I run the assay with just the core kit? 
No, both a core reagent kit and panel are needed to run this assay. There are different core kits depending on your sample type and concentration of purified RNA. Two types of panels are available – custom and focus. Discover everything you need to run a FirePlex miRNA assay.


6) What equipment do I need to run the assay?
The assay can be run on standard laboratory equipment. If you do not have the required equipment, our sample profiling service allows you to analyze miRNA with your own custom-designed panels, or with one of our convenient pre-designed focus panels.

Find out more about what you need to run the assay.

7) How long does the assay take to run?
Overall, the FirePlex miRNA assay takes 4–7 hours from sample to publication-ready data, depending on how many samples are processed in parallel and which cytometer is used for scanning.


8) What is the sensitivity of the assay?
The FirePlex miRNA assay detects targets from as little as 100 pg of total RNA.

With raw biofluid, the optimum input for the crude assay varies depending upon both sample input type and sample variability. You can use as little as 10 μL, but we recommend 20 μL as a starting point to test, adjusting input accordingly for future samples.

9) What is the reproducibility of the assay?
The reproducibility of the assay is very high: in our testing across four different samples for two replicates with 5 ng of starting total RNA, Pearson correlations were greater than 0.99. For more information please see the Technical Note.

10) How do I analyze the data? 
Interpretation and analysis of the assay results requires use of the FirePlex Analysis Workbench software, which you can download free of charge.

11) How does this technology differ from bead based assays?
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.

This patented hydrogel technology that has many advantages over bead based assays. These include

  • The entire hydrogel volume is used to capture your miRNA targets rather than just the surface of polystyrene beads, resulting in a much greater dynamic range.
  • Our hydrogel particles are non-fouling and bioinert, enabling detection straight from biofluids without the clumping often found if this is attempted with bead-based assays.
  • No specialized equipment is required for our assay, and readout is performed on standard flow cytometers.

12) Can I normalize my data using a synthetic target spiked into the serum or plasma samples?
If the synthetic target of choice is included within your custom or focused panel, the assay can detect that target and use it for normalization. However, in our experience normalizing using the geometric average of all detected targets is generally preferable, since synthetic spike-ins may partition within the sample in ways that are distinct from the endogenous miRNAs.

13) Which controls are included in the assay? 
X-control – the positive control included in the hybridisation buffer is a 21-nt long sequence that binds to the probe. There is also a blank negative control particle with no probe.

14) Should I include additional controls? 
Yes. Endogenous controls are strongly recommended as some miRNAs are known not to change their expression levels in samples/diseases studied.

15) What sample types are compatible with the assay?
Compatible sample types are purified RNA and crude biofluids. We have fully validated the assay with serum and plasma (including heparin plasma). We also have experience running the assay with other biofluids (including urine, cerebrospinal fluid, amniotic fluid and isolated exosomes) and other sample types. If you are interested in measuring miRNAs in samples other than RNA, plasma, or sera please contact us for the latest guidelines in sample input amounts and process modifications.


16) I want to run sample type that is not yet fully validated, can I do that?
Please contact technical@abcam.com to discuss the possibilities.


17) How much sample/starting material is required?
For biofluid, the optimum input for the crude assay varies depending upon both sample input type and sample variability. For plasma and sera we recommend 10–40 μL with 20 μL as a starting point to test, adjusting input accordingly for future samples.

When using purified RNA, use 1–5 ng total RNA.


18) How many samples can I run at once?
Our FirePlex miRNA assays are run in 96 well plates for high throughput analysis. We recommend running one 96 well plate at a time, but hundreds of samples can easily be analyzed over only a few days. Please remember that four wells per plate should be reserved for negative controls (water) and you may want to reserve other wells for positive controls (a reference RNA is included).


19) How should I prepare the sample?
Read our guidelines detailing RNA purification sample preparation for your FirePlex miRNA assay​.


RNA purification sample preparation guidelines

Read our guidelines detailing RNA purification sample preparation for your FirePlex® miRNA assay.​

​​

FirePlex miRNA Assay

For optimal results load between 0.1–5.0 ng of total RNA in a 25 µL volume per well, or add 40 µL plasma/serum to the digest step.

RNA isolation methods for different starting material are below:

Plasma or serum samples

For best results, store the sample at -80°C and limit the number of freeze/thaws that the samples undergo prior to quantification with the FirePlex miRNA Assay.

Isolated total RNA from cells and tissues

For best results, use any of the methods recommended for the FirePlex miRNA Assay.

Isolated total RNA from plasma or serum

For best results, isolate total RNA from plasma or serum using the TRIzol-LS®​ standard protocol as applied to 250 µL of sample, with 1 µL 15 mg/mL GlycoBlue™ added prior to precipitation. After precipitation with isopropanol, wash once with 75% ethanol then dry the pellet and resuspend in 50 µL RNase-free water. We suggest that 40 µL sample-equivalents be run (i.e. 8 µL of the 50 µL + 17 µL RNase-free water = 25 µL sample for assay).


Caution: For any column-based RNA isolation methodology, please confirm that the protocol you are using does not select against small RNAs. It is important to carefully follow the manufacturer's recommendations regarding ethanol concentration during wash and rinse steps to ensure that miRNAs are not excluded from the final product. Also, regardless of isolation method, it is important that the RNA be resuspended in RNase-free water or 1X TE to keep salt to a minimum.

Quantification of RNA from samples such as 250 µL plasma/serum will be impossible using absorbance-based technologies including Nanodrop and may be unreliable from more sensitive technologies such as Promega Quantus. Reliable profiling of miRNAs using the FirePlex miRNA Assay can be performed even on samples that are not adequately quantified.




FirePlex® is the new name for our Firefly assays.

FIREPLEX is a registered trademark in the United States and is an unregistered trademark elsewhere.


Equipment for FirePlex miRNA assays

A checklist of all the equipment necessary for our FirePlex miRNA assays.

Our FirePlex miRNA assays are designed with a simple workflow in mind, utilizing standard lab equipment for assay protocols and readout.

Get started with our equipment checklist of validated flow cytometers. In addition to our core reagent kit and your miRNA target panel, you will need:

  • Vacuum manifold
  • Shaking incubator
  • FirePlex analysis workbench software thermocycler
  • Standard flow cytometer

Vacuum manifold

Abcam’s vacuum manifold for 96-well filter plates (ab204067) is a cost-conscious alternative for simple filtration and collection in a 96-well filter plate format. The manifold is optimized for the efficient filtration of miRNA samples during the FirePlex miRNA assay protocol but can also be used for other samples.

Manifolds by other manufacturers are compatible with FirePlex assays, if the vacuum can be regulated at 5 psig.

Manifold specifications:

Material - ManifoldMaterial - gasketMaximum plate sizeVacuum gauge range
HDPEUrethane8.5 x 12.8 cm0 to -30 inHG
Filtrate only comes in contact with HDPE. HDPE is a common laboratory plastic with high chemcial resistance.
Several brands of plate meet this requirement, including Millipore Multiscreen and Pall AcroPrep plates. Nunc plates are not recommended.



Shaking incubator

FirePlex miRNA assays require four microplate shaking steps at different temperatures, with at least one shaker that can reach 60°C.

When repeating the assay multiple times, we recommend two different shakers for pre- and post- PCR steps. This is to minimize the risk of contamination of PCR amplicons between initial hybridization and labeling steps.

Compatible sample thermal shakers and incubators include:

  • Talboys Incubating Microplate Shaker
  • LabNet VorTemp 56 Shaking Incubator
  • VWR Symphony Incubating Microplate Shaker

Approved shakers have an orbital diameter of 3 mm and shake at 750 rpm. For shakers with a different orbital diameter, adjust the rpm according to the following formula:

rpm = (1687500/orbital diameter of shaker in mm)1⁄2

It is possible to modify other shakers to meet these specifications, but we do not recommend it.

Steps to be performed on a heated shaker cannot be performed on a thermocycler instead; optimal mixing is critical. 

Thermocycler

FirePlex miRNA assays require a thermocycler. While the assay is currently validated on the Eppendorf Mastercycler pro thermocycler, additional validations are pending, and multiple different models have been successfully tested.

When comparing results from a different thermocycler model, normalization methodology should be carefully assessed. Also note, absolute fluorescence may vary between thermocyclers; we recommend control runs on specific equipment to ensure that the observed fluorescence falls within the acceptable range.

Thermocyclers must be programmed as followed:

Number of cyclesTemperature (ºC)Time
193ºC15 s
27​93ºC5 s
59ºC15 s
72ºC60 s
693ºC5 s
63ºC15 s
72ºC60 s
172ºC5 min
194ºC4 min
14ºCForever

Supported cytometers

FirePlex miRNA assay particles are designed to be read on cytometers that have a blue (488 nm) laser with green, yellow and red detectors. FirePlex miRNA assays are optimized for the benchtop cytometers:

  • Millipore Guava easyCyteTM 6HT
  • Millipore Guava easyCyteTM 8HT
  • Millipore Guava easyCyteTM 12HT BD
  • AccuriTM C6
  • Life technologies Attune® Cytometer

Download the full list of validated flow cytometers

Please see the calibration and usage guides for these cytometers here.

For other machine configurations, please contact us at FirePlex.FAS@abcam.com


FirePlex miRNA assay controls

Recommended controls to optimize your results 

Experimental design is key to ensure high-quality and reliable miRNA expression data. The appropriate controls are essential to accurately subtract background signal, appropriately normalize data and assess inter-well variability.

FirePlex® miRNA assays include critical controls to ensure your results, including:

  • Multiple internal controls within each well
  • Negative control wells
  • Replicates


Internal controls

Each custom or focus panel includes:

Positive control

Each well contains positive control particles, which detect a miRNA-like target (the X-Control) present in the FirePlex Hybe Buffer. This control ensures successful assay implementation in each well.

Blank

Blank particles bear no probe, providing a baseline level of background fluorescence in each well.

Endogenous

Endogenous controls are small nucleolar RNAs or miRNAs expressed at consistent levels under a variety of conditions, to normalize the signal between sample types and treatments. Users may select one or more endogenous controls tailored to their research applications, with the help of FirePlex® Analysis Workbench analysis software. Our free and easy-to-use software reviews the expression of each target in each well of your experiments to identify the best endogenous control candidates for your selection.

All focus panels include at least three potential normalization targets.  If designing your own custom panel, we recommend including at least three of the targets below as potential normalizers. This will ensure targets that are detected at a high level in every sample to serve as endogenous controls.

Circulating assay potential normalizers: Human plasma/sera let-7d-5p, let-7g-5p, let-7i-5p, miR-29b- 5p

snoRNAs are unreliable controls in plasma or serum, miRNAs are recommended.

Remember, selecting targets NOT expected to change is just as critical as selecting targets that are expected to fluctuate.

Negative control wells

Negative control wells utilize a carrier buffer in place of a biological sample to measure background signal for each miRNA in the panel. Furthermore, multiple negative control wells allow for estimation of inter-well variability. We strongly recommend at least three negative controls per panel, every time an assay is performed.

If running the FirePlex miRNA (biofluid) assay for the first time, additional negative controls are recommended for the digest (by adding water to the lysis buffer) and for the assay as a whole (by adding water to the Hybe buffer in the first hybridization step).

Replicates

Replicates give statistical meaning to results by enabling the calculation of mean and standard deviation, for example. Replicates may be performed at the stage of sample preparation (biological) or assay (technical).

Biological

Biological replicates use the same conditions to treat and prepare samples from different sources to determine the biological variation within a population. For example, serum samples derived from three different mice injected with the same TNF inhibitor are biological replicates.

Technical

Technical replicates assay samples derived from the same source, multiple times to determine the reproducibility of the assay. Ideally, every assay should have technical replicates.


How does the FirePlex miRNA work?

The FirePlex miRNA assay allows profiling of up to 400 target miRNAs in the same well, directly from biofluids.

The FirePlex miRNA assay enables the profiling of up to 400 target miRNAs of choice simultaneously across tens or hundreds of samples. The assay can profile miRNAs directly from biofluids or from purified RNA, with assay readout on a standard flow cytometer. Find out more about the assay.

miRNA capture

Probes embedded within hydrogel particles have a miRNA binding site specific for a target miRNA. Flanking the miRNA binding site are universal adapter-binding regions, required for subsequent target amplification.

When particles are mixed with the sample (either crude biofluids after a digest step or purified RNA), target miRNAs bind to their specific probes. The hybridization buffer is optimized to keep only the right miRNAs bound to the probe, increasing the specificity of the assay.

Importantly, miRNA capture also acts as a purification step. After miRNA capture, particles are rinsed to remove unbound materials and potential inhibitors of subsequent steps. These include PCR inhibitors like heparin, which reduce sensitivity in other detection systems. Thus, FirePlex miRNA assays ideal for detecting miRNA targets directly from biofluid or low-quality RNA samples, regardless of purity.

Each well contains a mixture of particles specific to different target miRNAs, allowing for detection of multiple miRNA targets simultaneously. This enhances assay reproducibility, as all of the miRNAs quantified per well are exposed to exactly the same conditions throughout the assay.


End-labeling

Labeling mix containing universal adaptors and ligation enzymes is mixed with the particles, resulting in the ligation of adaptors on either side of the target miRNA to generate a fusion DNA-RNA-DNA molecule. This provides an additional level of specificity as miRNAs that are too long or too short do not ligate to the adapters.

Particles are rinsed and unligated adaptors, which are too short to remain on the probe through this step, are washed away.


Elution and universal amplification

Ligated miRNAs and adaptors are eluted from the probe, followed by a 60-min PCR program using primers specific for the universal adaptors. The reverse primer is labeled with biotin for later detection.


Recapture and report

After PCR amplification, samples are again mixed with particles to recapture amplified targets with the miRNA-specific probes.  The captured targets are then detected with a fluorescent reporter that binds to the biotin label. Particle fluorescence is measured by flow cytometry to quantitate target miRNAs.

The FirePlex miRNA assay protocol is simple and can be performed simultaneously across 96 wells, generating 6,528 data points in each run. The assay consists of (a) capture of selected miRNAs on encoded hydrogel particles, (b) ligation of specific adapters to each bound miRNA, (c) target amplification using a single, universal primer and (d) recapture of amplified targets onto the hydrogel particles. Assay readout is performed using a standard flow cytometer. The FCS fluorescence intensity data is decoded into target concentration in each well, and the data can be quickly visualized, manipulated and compared using the FirePlex Analysis Workbench software suite.


MicroRNA profiling methods: comparison of current technologies

At approximately 22 base pairs long, microRNAs (miRNAs) are a comparatively small target to experimentally verify and quantify in the lab. 

As the role of miRNAs in biological systems and their utility as biomarkers continues to expand, a number of techniques have been established for miRNA profiling.  However, technologies struggle to keep up with the growing bioinformatics and through-put demands of modern biomarker development research.

While some technologies strive to provide more comprehensive miRNA analysis, they struggle to deliver adequate sensitivity and specificity performance standards.  Meanwhile, more accurate and precise techniques tend to be too labor intensive for modern translational research.  FirePlex miRNA assays offer a new paradigm for multiplex miRNA analysis, simultaneously providing high-performance in a high-throughput format to deliver high-quality data for comprehensive miRNA profiling.

See how modern miRNA profiling methods compare and find out which method is most suited for your research goals in our at-a-glance table below.  Select each method for a more detailed look at benefits and drawbacks.

TechniqueWhen to useBenefitsDrawbacks
qPCR
  • Small scale experiments (1–2 samples or miRNAs)
  • Established protocols
  • High sensitivity and specificity
  • Labor intensive to scale
  • Requires quality miRNA annotation
miRNA arrays
  • Larger studies (has been used for up to 900 samples)
  • Established protocols
  • Purpose built analysis tools
  • Least quantitative
  • Requires quality miRNA annotation
RNA-seq
  • Discovery phase research
  • Whole genome analysis
  • Single base resolution
  • Does not require miRNA annotation
  • Less sensitive than qPCR
  • Requires most input material
  • Good degree of technical and bioinformatics skill necessary
Multiplex miRNA profiling
  • Multiplex studies (68 miRNAs, has previously been used for up to 600 samples)
  • High sensitivity and specificity
  • Straightforward data analysis
  • Can be used on crude biofluids
  • Requires standard lab equipment
  • Not ideal for small scale experiments of 1–2 samples
  • Requires quality miRNA annotation

miRNA qPCR assays

Quantitative real time PCR (qPCR) is one of the most popular techniques for validating and accurately quantifying miRNAs in relatively small experiments.

  • sensitive and quantitative with molar resolution
  • relatively inexpensive and flexible

This technique begins with the conversion of miRNA to cDNA. However, because the length of a miRNA molecule is comparable to that of a typical DNA primer, the molecule must be made longer by incorporating either a poly(A) tail or stem-loop structure.

The cDNA is then assayed as in a conventional qPCR experiment, with amplification initiated with a miRNA-specific primer and a stem-loop/poly(A) primer. The amplified product is detected with either SYBR® Green or a TaqMan® probe.

Limitations:

  • large experiments can be labor intensive due to low sample throughput
  • specificity issues may arise because, unlike conventional qPCR, only one flanking primer is specific to the miRNA—particularly problematic when using SYBR® Green
  • specificity issues can also arise when trying to distinguish miRNAs that differ by very few bases--due to the short template length, melting temperatures can be very low and very similar. Novel probes such as locked nucleic acids may mitigate specificity issues                (Vester et al., 2004)

​​

miRNA arrays

Conventional DNA oligonucleotide arrays are typically chosen for larger studies covering multiple miRNA targets.

  • relatively inexpensive way to measure hundreds of targets at once
  • mature technology with well-established protocols and purpose-built analysis tools available

Thousands of probes can be easily spotted on slides, or built up by photolithography, potentially enabling the parallel tracking of all known miRNAs. Arrays are probed by hybridizing fluorescently labeled DNA or RNA samples. The brightness of individual spots can infer relative changes in expression between samples.

Limitations:

  • Least quantitative of the three miRNA assay methods
  • As with qPCR, distinguishing similar sequences may be problematic, but may be mitigated by careful selection of control probes, stringent washing, and analysis.


RNA-seq

RNA-seq offers a high-throughput and sensitive option for miRNA profiling and is a particularly ideal discovery tool.

  • high-throughput capability of next-generation sequencing (NGS) platforms
  • capability to sample all miRNAs present in a sample, whether sequences are known or not
  • can distinguish closely related miRNAs and isoforms at single base resolution, by reading sequences directly

With these benefits, RNA-seq technologies can determine relative miRNA expression levels and, better yet, tagged libraries can be used for multiplexing.

The most popular NGS approach uses Illumina’s TruSeq kits to add sequencing adapters to an RNA library, which is then size fractionated to isolate miRNAs and run on one of Illumina's sequencing platforms. Illumina uses reversible dye- terminator sequencing to simultaneously sequence millions of library fragments in parallel.

Limitations:

  • Requires the most input material
  • Not as sensitive as qPCR
  • Library construction, especially amplification, is potentially a sizeable source of bias and requires a significant technical skill to undertake (Baker et al., 2010).
  • Data analysis can be relatively challenging, especially without a resident bioinformatician


MicroRNA profiling direct from biofluids, using FirePlex® particle technology

MicroRNA profiling direct from plasma or serum without RNA purification. Reduce variability, simplify workflow and conserve your sample.

The Multiplex Circulating miRNA Assay allows miRNA profiling direct from crude biofluids, including plasma, serum and exosomes, without the need for RNA purification. Find out how miRNA profiling direct from biofluids can benefit your experiment:

Reduced variability. Remove the unreliable RNA purification step in your sample preparation and get more consistent data.

Simplified workflow. Take less time from sample to data. RNA extraction adds significantly to the steps required to profile miRNAs across multiple samples. The Multiplex Circulating miRNA Assay with crude biofluids simplifies the workflow and cuts down assay time.

Smaller sample input. Profile miRNAs direct from as little as 10–40 μl crude biofluids. With the low abundance of miRNAs in biofluids, miRNA analysis typically requires a large quantity of biofluid for RNA extraction. Cut out this step to save your sample.

See the data for yourself

In this technical note, we demonstrate how this assay can be used for miRNA profiling with PCR sensitivity directly from as little as 20 µL of serum or plasma with no need for RNA purification, or from less than 100 pg of purified RNA. With our easy to use bioinformatics tools, we demonstrate that consistent miRNA profiles are obtained from plasma obtained using a variety of blood collection methods, including heparin.

Read the full technical note here 

miRNA profiling in crude digests versus extracted DNA. There was a close correlation between miRNA expression profiles obtained from purified miRNA and those obtained from crude serum.



References

  • Chapin SC, Pregibon DC and Doyle PS (2011). Rapid mircoRNA profiling on encoded gel microparticles. Angew Chem Int Ed, 50, 2289-93.
  • Chapin SC, Pregibon DC and Doyle PS (2009). High-throughput flow alignment of barcoded hydrogel microparticles. Lab Chip, 9, 3100-9.
  • Dendukuri D, Pregibon DC, Collins J, Hatton TA and Doyle PS (2006). Continuous flow lithography for high-throughput microparticle synthesis. Nat Mater, 5, 365-9.
  • Pregibon DC and Doyle PS (2009). Optimization of encoded hydrogel particles for nucleic acid quantification. Anal Chem, 81, 4873-81.
  • Prefibon DC, Toner M and Doyle PS (2007). Multifunctional encoded particles for high-throughput biomolecule analysis. Science​, 315, 1393-96.

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FirePlex miRNA Disease Focus Panels

Get a head start on your translational research goals with our pre-designed disease focus panels

Efficiently survey dozens of miRNAs most relevant to specific disease categories with our multiplex circulating miRNA disease focus panels.  Easily, quickly and comprehensively profile miRNAs directly from biofluids (serum, plasma, exosome or purified RNA) to identify and validate important diagnostic, prognostic and treatment-related biomarkers of disease.

Each disease-specific panel consists of 68 pre-selected miRNAs, curated from the current peer-reviewed literature for their involvement in the disease category of interest.  These include miRNA differentially expressed between normal and diseased states, in whole blood, plasma, serum, peripheral blood mononuclear cells (PBMCs) or isolated exosomes. Panels include general markers of each disease category, as well as markers of specific disease subsets and pathological states, providing full coverage of biomarker candidates for each field of interest.

Disease panels are specifically designed for translational value, with most of the chosen miRNA targets identical in mouse, rat and human systems (Panel dependent, ranging from 55 to 61 out of 68 total).  Accelerate your research from discovery to clinical stages with easy grab-and-go FirePlex disease focus panels.

Cardiology Panel (ab218368)

  • Markers specific to certain cardiovascular states or treatment responses

View the complete miRNA list and supporting references


Immunology Panel (ab218369)

  • Markers specific to certain autoimmune diseases and blood-based cancers

View the complete miRNA list and supporting references


Oncology Panel (ab218367)

  • Markers specific to certain cancer types 

View the complete miRNA list and supporting references


Neurology Panel (ab218371)

  • Markers specific to certain nervous system diseases, psychiatric states and CNS cancers

View the complete miRNA list and supporting references


Liver Tox Panel (ab218370)

  • Markers specific to certain liver states, liver toxicity, hepatocellular carcinoma or treatment responses

View the complete miRNA list and supporting references


Each disease focus panel also includes:

  • Internal controls to validate assay performance​
  • Two - four stably expressed miRNAs to help with data normalization

See more about recommended controls for FirePlex miRNA immunoassays

See additional FirePlex miRNA Toxicity Screening Panels

Haven't found the miRNAs you are looking for? Design a custom panel

FirePlex® is a registered trade mark in the United States and is an unregistered trade mark elsewhere. 






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