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The Multiplex Circulating miRNA Assay enables the profiling of up to 68 target miRNAs of choice simultaneously across tens or hundreds of samples. The assay can be used to profile miRNAs directly from crude biofluids or from purified RNA, with assay readout on a standard flow cytometer. Find out more about the assay.
Figure 1. Molecular workflow for the Multiplex Circulating miRNA Assay.
Probes embedded within hydrogel particles have an miRNA binding site specific for a particular target miRNA. Flanking the miRNA binding site on the probes are universal adapter-binding regions required for subsequent amplification.
When particles are mixed with sample (either crude biofluids after a digest step or purified RNA), target miRNAs bind to their specific probes. The hybridization buffer used for this step is optimized to keep only the right miRNAs bound to the probe, increasing the specificity of the assay. After miRNA capture, particles are rinsed to remove unbound materials and remove potential inhibitors of subsequent steps, such as heparin.
A mixture of particles specific to different target miRNAs is used in an experiment, meaning that multiple miRNAs are targeted within a single well. This greatly contributes to assay reproducibility, as all of the miRNAs quantified per well are exposed to the same conditions throughout the assay.
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 and a 60 minute PCR program is performed using primers specific for the universal adaptors. The reverse primer is labeled with biotin, allowing reporting of the target miRNAs at a later step.
Recapture and report
After amplification, samples are mixed with particles once more to be recaptured by miRNA-specific probes. A fluorescent reporter is added that binds to the biotin incorporated during amplification. Fluorescence is then measured on the particles by flow cytometry.
Figure 2. Protocol workflow for the Multiplex Circulating miRNA Assay.