The MACSPlex miRNA Kit – Cancer has been developed for the simultaneous flow cytometric detection of 39 miRNAs that are known to be deregulated in different cancer types in a single sample.
MACSPlex miRNA Kit analysis is based on MACSPlex (MPx) miRNA Cancer Beads, which display defined fluorescence properties and can be identified using standard flow cytometry techniques. Within an array MPx miRNA Cancer Beads contain a cocktail of 39 fluorescently labeled bead populations, each coupled with a specific oligonucleotides.
After enzymatic labeling of the samples with Vio®
667, the samples are incubated with MPx miRNA Cancer Beads and the labeled miRNAs hybridize to the specific oligonucleotides. The hybridized beads can be analyzed based on the fluorescence characteristics of both the MPx miRNA Cancer Beads and the hybridized miRNAs. They can be distinguished by different fluorescence intensities detected in the B1 and B2 channel of the MACSQuant®
Analyzer and MACSQuant Analyzer 10 or the FITC and PE channel of other flow cytometers.
The starting material for detection of miRNAs can either be total RNA or size-fractionated total RNA. Depending on the isolation method, small RNAs may get lost. Thus, we recommend using total RNA. Total RNA of the samples of interest is labelled with MPx miRNA Enzyme 1 and MPx miRNA Enzyme 2. After the 2.5-hour labeling procedure the MPx miRNA Cancer Beads and the MPx miRNA Hybridization Buffer are added to the samples. During a 3-hour incubation period, the miRNAs hybridize to the MPx miRNA Cancer Beads. The median of the Vio 667 fluorescence (R1/APC channel) of each hybridized bead population is used for the calculation of specific miRNA expression levels between samples.