Ultra-sensitive detection of nucleic acid mutations for research and clinical use

What is superRCA?

superRCA is an ultra-sensitive and highly specific molecular amplification technology. It is used to detect very small amounts of DNA sequence variants, like cancer mutations, in patient tissue and blood samples.

superRCA offers effective detection of multiple targets simultaneously – so called multiplexing. The assay can be performed in most hematology laboratories with existing equipment, using well-established flow cytometry for read-out, enabling more accessible testing with shorter response times.

The convenient, non-intrusive liquid biopsy-format enables higher frequency of testing for improved patient monitoring and earlier detection of relapse.

In the clinic, superRCA can be used by the practicing clinician to monitor disease progress for adjusted treatment and personalized medicine. Our first application is in Acute Myeloid Leukemia (AML), offering single and multiplex testing for recurring tumor-specific mutations.

For researchers and pharmaceutical developers, our RUO kits offer a convenient and efficient toolbox for investigating numerous therapeutic areas to design companion diagnostics and advance personalized medicine.

superRCA offers a personal diagnostics platform for clinicians while providing an open learning system for researchers, partners and collaborations that allows new discoveries.

Ultra-sensitivity – detects 1 mutation in 100,000 wild types

superRCA can detect 1 mutation out of a 100 000 wild-type DNA molecules.

The unprecedented sensitivity of superRCA is achieved by two consecutive Rolling Circle Amplification (RCA) reactions. A first standard RCA step is followed directly by a subsequent “in situPadlock Probing and RCA step. As a result the target region is genotyped with high specificity, enumerated with higher precision.

superRCA method

An indepth introduction to superRCA

superRCA utilizes Rolling Circle Amplification (RCA) and Padlock probes in a novel way to achieve highly specific, ultra-sensitive detection of nucleic sequences. The method produces a relatively large, self-constrained structure – a superRCA structure – that can be directly analyzed by microscopy or automated using flow cytometry without the need for partitioning.

Flow cytometry with fluorescent labelling ensures that multiple targets can be analyzed simultaneously. As a result, the platform is extremely effective for patient-near, fast and cost-effective patient monitoring as well as companion diagnostics and pharmaceutical development .

DNA is extracted from the sample, either whole blood, bone marrow or tissue.

The DNA sequences of interest, known to be mutated in a patient’s malignant cells, are first enriched by a limited pre-PCR (~10 cycle) amplification.

The enriched sample then undergoes a ligase-mediated circularization of one strand.

The circularized strands containing the target region are then amplified by the first Rolling Circle Amplification  (RCA) step.

This is followed by Padlock Probe ligation and a highly-specific, second RCA step. During this step, the second RCA encircles the first RCA product to form large superRCA structures that can be analyze by flow cytometry.

The superRCA products can be scored as mutant- or wildtype-specific using fluorophore-labeled hybridization probes and recorded as individual, brightly fluorescent objects in a standard flow cytometer. For multiplex assays, analysis is similarly done by using multiple fluorophores and wavelengths.

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Why superRCA makes a difference

  • Unprecedented ultra-sensitivity of 1 mutation in 100 000 wild types due to the novel combination of the extremely high specificity of Padlock probes and RCA
  • Meets or exceeds currently available technologies like ddPCR, NGS and traditional liquid biopsy assays by 10-100 times
  • Outstanding performance on High GC% targets. Assessment of high GC content with the same sensitivity as low GC content – with minimum bias.
  • High multiplexity creates opportunities for effective research, monitoring therapies and drug resistance, sensitive multiplex detection of mutations for treatment selection (companion diagnostics), quantitative measurement for patient follow up and clinical phase drug development
  • Convenient and reliable analysis by well-established flow cytometry for speed by patient-near testing.
  • Other read-out and analysis methods including fluorescence microscopy
  • Speed and cost-effectiveness at high-throughput.
  • An integrated sample-to-answer system with robotic automation using standard lab equipment and well-established instrumentation, minimizes manual handling and is highly amenable to clinical practice
  • Currently available as research use only kit or as a customized service

The flexibility of multiplexing superRCA 

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.

Rarity Bioscience Parallelplex

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.  

Rarity Bioscience Ratiolabeling

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.  

Rarity Bioscience Colorplex

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.  

Rarity Bioscience Comboplex

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.