biostudies-literatureRajagopal ACalifornia Institute of Technology1053https://www.ebi.ac.uk/biostudies/studies/S-EPMC6355831biostudies-literatureUnknown9(1)The real time polymerase chain reaction (rtPCR) is an essential method for detecting nucleic acids that has a wide range of clinical and research applications. Current multiplexed rtPCR is capable of detecting four to six nucleic acid targets in a single sample. However, advances in clinical medicine are driving the need to measure many more targets at once. We demonstrate a novel method which significantly increases the multiplexing capability of any existing rtPCR instrument without new hardware, software, or chemistry. The technique works by varying the relative TaqMan probe concentrations amongst targets that are measured in a single fluorometric channel. Our fluorescent amplitude modulation method generates a unique rtPCR signature for every combination of targets present in a reactioScientific reportsSignificant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation.PMC6355831CIT2-Rajagopal-2014Shin CTombrello TATsongalis GJMenge KYurk DFraser SRajagopal AJacky LfalseSignificant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation.The real time polymerase chain reaction (rtPCR) is an essential method for detecting nucleic acids that has a wide range of clinical and research applications. Current multiplexed rtPCR is capable of detecting four to six nucleic acid targets in a single sample. However, advances in clinical medicine are driving the need to measure many more targets at once. We demonstrate a novel method which significantly increases the multiplexing capability of any existing rtPCR instrument without new hardware, software, or chemistry. The technique works by varying the relative TaqMan probe concentrations amongst targets that are measured in a single fluorometric channel. Our fluorescent amplitude modulation method generates a unique rtPCR signature for every combination of targets present in a reactio2019-01-01T00:00:00Z2019 Jan2025-04-20T02:11:08.632Z2019-03-26T22:50:36ZS-EPMC63558313070533310.1038/s41598-018-37732-y