dPCR Copy Number Variation (CNV) Probe Assays

• More than 200 wet-lab-validated assays and a custom tool for in-silico assay design
• Dye selection enables multiplexing of up to 5 targets per reaction
• Gene of interest, reference and centromeric reference assays available
• Ready-to-use assays have been validated for a range of samples, such as FFPE, cell lines and gDNA
• Simple and fast dPCR workflow on the QIAcuity
• Convenient copy number data analysis using the QIAcuity Software Suite

dPCR CNV Probe Assays enable specific, accurate, reproducible and easy-to-interpret copy number change analysis for individual genes or regions of interest. Assays for more than 200 targets have been PCR wet-lab-validated and are flagged as such in the particular assay description.

Additionally, a custom design tool is available to address assay needs outside of the wet-lab-validated assay offering.

The dPCR CNV Probe Assays are available in a tube format as a ready-to-use, 20x-concentrated assay. The tube contains the primer pair and a hydrolysis probe for use with the QIAcuity Probe PCR Kit (DNA targets), the QIAcuity Digital PCR System and QIAcuity Nanoplates .

Each assay includes the choice of five different hydrolysis probes (fluorophores): FAM, HEX, ROX, Atto 550 and Cy5. This flexibility allows the mixing and matching of assays for multiplex analysis of up to five targets in a single dPCR reaction.

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Outstanding assay performance

The dPCR CNV Probe Assays exhibit excellent product amplification and separation between the negative and positive partition clusters in a duplex analysis using two different gDNA template concentrations, (see 1D and 2D scatterplots of duplex reactions for KRAS and TERT assays). The results indicate that accurate analysis of CNVs can be performed even at low concentrations of gDNA input. For minimum and maximum loading amounts into 8.5k nanoplates, please check the QIAcuity user manual extension.

Precise and reproducible copy number analysis using digital PCR

The highly reproducible QIAcuity dPCR system and the dPCR CNV Probe Assays allow CNV analysis without the need of replicates, increasing the sample throughput in a cost-effective manner. Duplex analysis of a gDNA sample resulted in the expected number of copies per genome for all replicates (see Accurate and reproducible copy number analysis in a duplex reaction). In a precision test analyzing a gDNA dilution series, the assays exhibit excellet linear distribution and cluster separation (see Linear distribution in a dilution series precision test).

Multiplex copy number analysis by dPCR

Multiplex analysis with the dPCR CNV Probe Assays increases throughput and significantly reduces analysis costs per target. The ability to order these assays with different dyes (FAM, HEX, ROX, Atto 550 and Cy5) enables a flexible experimental design and multiplex analysis of up to 5 targets simultaneously in a single reaction. The dPCR CNV Probe Assays show an excellent amplification and positive and negative cluster separations for 5 assays multiplexed in a single reaction (see CNV detection in a 5-plex reaction).

All dPCR CNV Probe Assays are designed for unique regions of the human genome. The dPCR CNV Probe Gene of Interest Assays target highly studied cancer and cancer-related genes. The dPCR CNV Probe Reference Assays and dPCR CNV Probe Centromeric Reference Assays include a variety of references with different copy numbers per genome, making it possible to select the optimal normalization references for each analysis. Depending on the experimental conditions, multiple GOI and reference assays can be included in the reactions for accurate calculation of the copy number of the gene or target of interest.

The dPCR Copy Number Probe Assays are ready-to-use: just add the individual assay mixes to the DNA sample and Mastermix from the QIAcuity Probe PCR Kit, load into the QIAcuity Nanoplate and start the run on the QIAcuity instrument. Each assay is based on an end-point PCR amplification of a specific region of genes across the human genome. The amplified product is detected using target-specific fluorescent hydrolysis probes, which help to ensure high assay specificity.

The principle of the dPCR reaction in the nanoplates is described here.

The experimental setup procedure is simple and straightforward and can be performed in any laboratory that uses the QIAcuity dPCR instrument. For a more precise copy number analysis, we recommend following the genomic DNA isolation from the sample with a restriction enzyme digestion. Restriction digestion is not required for highly fragmented DNA (e.g., FFPE DNA or circulating DNA) or cDNA. To perform the restriction digestion directly in the reaction mix, include the recommended restriction enzyme during the reaction setup. It is critical to avoid using restriction enzymes that cut within the target amplicon region.

The fragmented template is then mixed with the ready-to-use QIAcuity Probe PCR Kit mastermix and the dPCR CNV Probe Assay, and this mixture is aliquoted into each well of the dPCR preplate. The reaction mix is then mixed well via pipetting and is transferred into the wells of a QIAcuity Nanoplate. Each gene or target of interest and each reference assay can be analyzed in a single well in a multiplex reaction by combining them with different fluorophores.

After loading and sealing the nanoplate the recommended cycling program is run on the QIAcuity instrument. The resulting copies per microliter are shown in the absolute quantification analysis type of the QIAcuity Software Suite. For the calculation of copy number per genome, select the copy number data analysis type in the QIAcuity Software Suite, and define the nanoplate wells for the gene/region of interest or the normalization reference. Depending on the cycling protocol, the results of the dPCR run can be analyzed in the QIAcuity Software Suite after ~2 hours.

dPCR CNV Probe Assays are highly suited for accurately detecting copy number alterations or variations at individual loci using DNA from fresh, frozen or fixed samples.