Our multiplex circulating miRNA assay has 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 below to help you get started.
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 multiplex circulating miRNA assay protocol, but can also be used for other samples.
Manifolds by other manufacturers are compatible with Firefly® assays, if the vacuum can be regulated at 5 psig.
|Material - Manifold||Material - gasket||Maximum plate size||Vacuum gauge range|
|HDPE||Urethane||8.5 x 12.8 cm||0 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.|
The multiplex circulating miRNA assay requires four microplate shaking steps at different temperatures. The assay requires at least one shaker that can reach 60°C. If the multiplex circulating 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 multiplex circulating microRNA assays include:
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.
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 multiplex circulating miRNA assay has been optimized to work with include:
Calibration and usage guides for these cytometers are available here:
For other machine configurations, please contact us.
For the multiplex circulating 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 cycles||Temperature (ºC)||Time|
FireflyTM Analysis Workbench software
The Firefly Analysis Workbench is a powerful analysis tool that processes raw cytometer data from the Multiplex miRNA Assays into publication-ready charts and heatmaps.
Firefly® is a registered trade mark in the United States and is registered as a European Union Trade Mark.