Digital PCR (dPCR) workflows increasingly demand higher levels of multiplexing to extract more actionable data from limited nucleic acid samples.
Traditional low-plex approaches can consume valuable sample, extend turnaround times and introduce variability through repeated handling – especially when copy number changes or closely related targets are involved.
This application note explores how six-channel dPCR multiplexing supports simultaneous detection of multiple RNA or DNA targets, enabling clear signal separation and reliable quantification across channels.
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The detection of multiple targets in the same dPCR reaction can conserve sample, shorten turnaround times, reduce pipetting errors, and maximize the data obtained from each run. The Digital LightCycler® dPCR System can detect as many as 6 separate RNA or DNA targets in a single reaction while utilizing a separate channel as a process control. Detect up to 6 unique targets in a single reaction B2M B2M In the examples below, six separate genes are assayed simultaneously with different dyes, on six separate channels. Of these targets, four are frequently amplified in cancer, and two serve as a references for the nonamplified copy number (Table 1). HOGA-1 (Atto425) MET (FAM) FGFR2 (Cy5) EGFR EGFR (Cy5.5) MET HOG1A EGFR MET Figure 1. 2D scatter plots showing robust performance, clear separation of signals, and lack of cross-channel interference. In each panel, data are shown for two channels; red denotes empty (negative) partitions and green denotes partitions containing both signals. Assay input: 50 ng of Clontech gDNA. B2M (HEX) FGFR1 (Texas Red) Table 1. Channel assignment for targets of interest and reference targets of the example 6-plex assay. See Figure 4 for a complete list of compatible dyes. Gene of interest* HOGA1 MET B2M FGFR1 FGFR2 EGFR Optical Channel 1 2 3 4 Dye Atto425 FAM Alteration in cancer Not expected (reference) Amplification HEX Not expected (reference) FGFR2 Y X FGFR2 FGFR1 Texas Red 5 6 Cy5 Cy5.5 Amplification Amplification Amplification *Genes are: B2M, β2-microglobulin; EGFR, epidermal growth factor receptor; FAM, fluorescein amidite; FGFR1/2, fibroblast growth factor receptor 1/2; HEX, hexachloro-fluorescein; HOGA1, 4-hydroxy-2-oxoglutarate aldolase 1; MET, mesenchymal-epithelial transition. Z Figure 2. 3D scatter plots, each showing signal from 3 channels. Each “wall” in these charts shows signal from 2 targets, and is read similarly to the 2D charts shown in Figure 1. In each chart, the small, dark-blue cluster (blue circle) where 3 axes meet represents partitions that are negative for all 3 signals, and the light purple cluster (purple oval) that appears closest represents partitions that are positive for all 3 targets. FGFR1 HOG1AChannel 1 Atto425 - HOGA1 Channel 2 FAM - MET Channel 3 HEX - B2M Channel 4 Texas Red - FGFR1 Channel 5 Cy5 - FGFR2 Channel 6 Cy5.5 - EGFR Figure 3. 1D scatter plots of individual channels from the assays shown in Figures 1 and 2. See Figure 4 for additional dyes that are compatible with each channel. Compatible optical dyes Channel 1 Coumarin Cyan500 Atto425 Channel 2 FAM Channel 3 HEX VIC Figure 4. Dyes compatible with the Digital LightCycler® dPCR System. Channel 4 LC610 TexRed CFR610 Channel 5 CY5 JA270 LC640 Channel 6 CY5.5 Learn more about how the Digital LightCycler® dPCR System supports discriminative multiplexing for up to 6 targets on 6 separate channels at go.roche.com/dpcr or by scanning the QR code. The Digital LightCycler® is a Class II US IVD instrument. © 2023 Roche Sequencing and Life Science. All rights reserved. MC-US-13487 A726 07/23
