All tags Multiplex miRNA assays FirePlex® miRNA assays FAQ

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​.



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