MicroRNA profiling

As microRNA (miRNA)-based research shifts from discovery of new miRNAs to elucidating their biological roles and their utility as biomarkers, robust methods are needed to profile large sample cohorts.

To address this need, SmartRNAplex assays provide a method for multiplexed miRNA analysis of up to 68 targets across as many as 96 samples. The SmartRNAplex assay is completely customisable - researchers may design panels to detect any combination of miRNAs annotated in miRbase (from any species).

Multiplexing is accomplished by using encoded hydrogel Firefly particles. Each Firefly particle bears a unique ‘barcode’ that identifies the miRNA species for which that particle bears a complementary binding sequence. Including hands-on time, the SmartRNAplex assay takes 3.5 to 4.5 hours from samples to data, depending on how many samples are processed in parallel (Figure 1).

Figure 1: The SmartRNAplex assay takes 3.5 to 4.5 hours from samples to data.

Unlike other systems that rely on glass or polystyrene substrates, Firefly particles are composed of bio-inert poly (ethylene glycol) hydrogels. This unique substrate provides solution-like thermodynamics for optimal sensitivity and specificity, and enhanced capacity for three-dimensional target capture leading to a greater dynamic range. This substrate, coupled with the SmartRNAplex post-hybridisation labelling method, makes the platform ideal for detection in crude samples eliminating the need for RNA purification. Additionally, bound targets can be labelled on either the 3′ or 5′ end in order to discriminate mature miRNAs from precursor species.

Unlike other labelling schemes, the SmartRNAplex assay labels targets after they have been captured by miRNA-specific probes embedded in the hydrogel particles. Probes have two binding sites: one site binds a specific miRNA, and the other site binds universal adapter sequence used for labelling. When a miRNA target is captured on its corresponding probe, a universal adapter is attached to that miRNA via ligation. After addition of a reporter species, this binding event is detected via fluorescence. The level of fluorescence is quantitative, providing an accurate indication of target levels in a given sample.

Final assay readout can be performed using a standard flow cytometer. The minimum configuration of the system, given the current particle design, is a single blue laser (~488 nm) for excitation with green (~525 nm), yellow (~580 nm), and red (~690 nm) detectors. Firefly particles are designed to appear to the cytometers as a series of closely spaced cells and each particle is recorded as multiple sequential events.  In order to interpret the collected data, we developed Firefly software to analyse FCS data generated with the SmartRNAplex assay. The software parses through the events contained within the FCS file(s) and regroups them into particle information, with barcode data and target levels, in a matter of seconds. These data are presented in plots showing the heat map and target quantification for a given sample.

In conclusion: the SmartRNAplex assay provides a means to profile miRNA in any species across a broad range of starting material. The multiplex assay is completely customisable for detection of up to 68 miRNA targets per sample with a rapid workflow. This enables the ability to easily discover which miRNA targets are relevant to one’s research without the laborious disadvantages of traditional methods.

Merck Pty Limited
www.merckmillipore.com