The SuperRCA technology has intrinsic properties that allows for Multiplexing in a number of different ways based on application and user need. Multiplexing, in any form, means to be able to analyze a single sample for several sequences variations.
For all SuperRCA assays, the first step is an initial pre-amplification, were an assay specific amplicon is used for multiplexed target enrichment. This is undergoing approximately ten cycles of PCR, meaning enough to increase you initial sample to ensure statistical detection, but low enough to avoid PCR induced errors affecting the specificity. This step is comparable with library prep for sequencing.
Once the library prep is completed, the sample can undergo the SuperRCA incubation in a few different ways depending on the type of multiplex readout.
For Parallel-Plex, the pre-amplified products are split into several wells and incubated with different padlock targets per well, meaning that each well is then analyzed for its individual target mutation. This can be done straight forward with only two colors for mutant and wildtype in each well.
If high throughput and low running costs is important, we use Ratio-Labeling Multiplex. Here the pre-amplified products remain in one single well, and different ratios of the two fluorophores are added to the different target probes. During the flow-cytometry read-out, presence of the different mutations will occur as cluster based on the specific ratios, and can be gated accordingly. This enables multiplex analysis while still only using two colors.
For even higher throughput and cost efficiency, one can use Multicolor Multiplex, where different fluorophores are attached to each padlock probe, allowing simultaneous readout of multiple targets. This is limited to the number of lasers in the flow cytometer as well as the choice of dyes, and for additional expansion, multiple colors can be combined with ratio labeling to reach maximum multiplex.
The final option that can be combined with any of the above is Comboplex, where the same fluorophore is attached to multiple mutant probes, meaning that the readout will provide an answer if any of the mutations is present in the sample, but not particularly which one. This can be used for long term monitoring and/or in combination with a secondary drop-down assay in case sample is positive.
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