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. 2023 Aug 21;83(21):3636–3649. doi: 10.1158/0008-5472.CAN-22-3402

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©2023 The Authors; Published by the American Association for Cancer Research

This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.

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Figure 4. Performance evaluation of multi-msqPCR. A, Comparison between the multi-msqPCR and uniplex-msqPCR assays. The multi-msqPCR assay produced higher ΔCq values than uniplex-msqPCR assays [6.13 (ΔCq multiple hypermethylated sites) versus 1.44 (ΔCq cg11754974), 1.97 (ΔCq cg24905336), 0.47 (ΔCq cg22678228; left) and 4.43 (ΔCq multiple hypomethylated sites) versus 2.18 (ΔCq cg26026551), 1.79 (ΔCq cg00227790; right)]. B, Assessment of the analytic sensitivity of the multi-msqPCR assay. Primer probe sets for multiple hypermethylated sites (left) and multiple hypomethylated sites (right) were tested for two technical replicates with fold dilutions of 50 ng bisulfite-converted DNA. The multi-msqPCR assay detected tumor DNA signals with as little as 0.1%. ΔCq = (CqACTB− Cqbiomarker). — Performance evaluation of multi-msqPCR. A, Comparison between the multi-msqPCR and uniplex-msqPCR assays. The multi-msqPCR assay produced higher ΔCq values than uniplex-msqPCR assays [6.13 (ΔCq _{multiple hypermethylated sites}) versus 1.44 (ΔCq _cg11754974), 1.97 (ΔCq _cg24905336), 0.47 (ΔCq _cg22678228; left) and 4.43 (ΔCq _{multiple hypomethylated sites}) versus 2.18 (ΔCq _cg26026551), 1.79 (ΔCq _cg00227790; right)]. B, Assessment of the analytic sensitivity of the multi-msqPCR assay. Primer probe sets for multiple hypermethylated sites (left) and multiple hypomethylated sites (right) were tested for two technical replicates with fold dilutions of 50 ng bisulfite-converted DNA. The multi-msqPCR assay detected tumor DNA signals with as little as 0.1%. ΔCq = (Cq_ACTB− Cq_biomarker).