All tags Multiplex miRNA assays What equipment do you need to run the FirePlex® miRNA assay?

What equipment do you need to run the FirePlex® miRNA assay?

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

Our FirePlex miRNA assays have been designed with a simple workflow in mind. You can read the assay on one of a number of validated flow cytometers and only a few other items of standard lab equipment is required to run the assays.

Use our checklist of required equipment to help you get started:

  • Core reagent kit
  • Partcle panel
  • Vacuum manifold
  • Shaking incubator
  • FirePlex analysis workbench software
  • Thermocycler

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Vacuum manifold

Our vacuum manifold for 96-well filter plates (ab204067) is a cost-conscious alternative for simple filtration and filtrate collection in a 96-well filter plate. 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.

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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

The FirePlex miRNA Assay requires four microplate shaking steps at different temperatures. The assay requires at least one shaker that can reach 60°C. If the FirePlex miRNA assay is to be repeated multiple times, we recommend having two shakers; one for the pre-PCR steps and one for the post-PCR steps. This is because it is very important that PCR amplicons do not contaminate initial hybridization and labeling steps.

Sample thermal shakers and incubators compatible with the FirePlex miRNA assays 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)½

It is possible that you can 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.


Supported cytometers

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

  • Millipore Guava easyCyte™ 6HT
  • Millipore Guava easyCyte™ 8HT
  • Millipore Guava easyCyte™ 12HT
  • BD Accuri™ C6
  • Life technologies Attune® Cytometer

Calibration and usage guides for these cytometers are available here:

For other machine configurations, please contact us.


Thermocycler

For the FirePlex miRNA assay you will need to use a thermocycler. This assay has been successfully tested on multiple models of thermocycler.

Absolute fluorescence may vary between thermocyclers and we recommend that users perform control runs on their specific equipment to ensure that the observed fluorescence fits within an acceptable range.

Care should be taken with normalization methodology when comparing results from use of a different thermocycler model.

This assay is currently validated on the Eppendorf Mastercycler pro thermocycler, additional validations are pending.

Thermocyclers must be programmed as followed:

Number of cyclesTemperature (ºC)Time
193ºC15 s
2793ºC5 s
59ºC15 s
72ºC60 s
693ºC5 s
63ºC15 s
72ºC60 s
172ºC5 min
194ºC4 min
14ºCForever


FirePlex Analysis Workbench software

The FirePlex Analysis Workbench is a powerful analysis tool that processes raw cytometer data from the FirePlex miRNA Assays into publication-ready charts and heatmaps.

Find out more or download the software here.

FirePlex® ​ is the new name for our Firefly assays.