Accelerated qPCR Assays With Enhanced Multiplexing Technology

Average ∆Cq Sample input (ng/reaction) Dye 6 Dye 5 Dye 4 Dye 3 Dye 2 Dye 1 Target 0.1 1 10 100 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 Dye 1 Dye 2 Dye 3 Dye 4 Dye 5 Dye 6 Dye 1 Dye 2 Dye 3 Dye 4 Dye 5 Dye 6 Dye 1 Dye 2 Dye 3 Dye 4 Dye 5 Dye 6 Dye 1 Dye 2 Dye 3 Dye 4 Dye 5 Dye 6 TaqMan QSY and QSY2 probes Multiplex better with the newest additions to the TaqMan portfolio Product overview Assay developers are under constant pressure to save time and reduce costs, while also assuring accuracy and delivering reproducible results. This can be accomplished by maximizing the number of targets per sample by multiplexing. With new Applied Biosystems™ TaqMan™ QSY™2 probes, users can now multiplex up to six targets in a single real-time PCR (qPCR) reaction with higher sensitivity, greater dynamic range, and better assay performance retention when scaling from 1-plex to 6-plex. These probes will enable users to speed up optimization of their multiplex assays and leverage the full capabilities of their qPCR instruments with higher multiplexing. Equipped with the cyanine 5 and cyanine 5.5 long-wavelength dyes, the TaqMan QSY2-quenched probes enable lower background and greater signal-to-noise ratios while detecting targets in the far-red spectrum. This wavelength coverage complements that of the Applied Biosystems™ TaqMan™ QSY™ probes that are available with Applied Biosystems™ FAM™, VIC™, ABY™, and JUN™ reporter dyes. TaqMan QSY and QSY2 probes may be configured and ordered online via the Applied Biosystems™ Custom TaqMan™ Probes tool. Head-to-head performance—TaqMan QSY and QSY2 probes vs. IDT probes Two 6-plex combinations of TaqMan QSY and QSY2 probes were run against Iowa Black™ single-quenched probe, and Zen™/Iowa Black™ and TAO™/Iowa Black™ double-quenched probes (Integrated DNA Technologies). All other variables were controlled. Double-quenched probes were selected from Integrated DNA Technologies (IDT™) products where available. Each probe was also run in a single-plex assay on the same plate. The results from testing show that TaqMan QSY and QSY2 probes are more sensitive, have greater dynamic range, and better retain single-plex performance when scaling to a 6-plex assay, than their IDT counterparts (Figures 1–5). Sensitivity Sensitivity is the ability to detect targets at low concentrations. When comparing two probes targeting a controlled input, an earlier Cq indicates greater sensitivity. Figure 1 shows the average ΔCq between the IDT and TaqMan probes. A positive average ΔCq value indicates a later Cq for the IDT probe and earlier Cq for the TaqMan probe—and therefore greater sensitivity of the TaqMan probe. Figure 1. Average ΔCq between TaqMan and IDT probes in six-plex assays. TaqMan probes demonstrate higher sensitivity and earlier detection across multiple sample inputs, as indicated by earlier average Cq values and positive average ΔCq values. Average ΔCq = (average Cq values using IDT probes)–(average Cq values using TaqMan probes). qPCR probes 70% (17/24) of the time, TaqMan probes have an earlier average Cq than IDT probes when run in 6-plex. On average, the Cq values of TaqMan probes are 2.7 cycles earlier than those of IDT probes when run in a 6-plex assay. Dynamic range A greater dynamic range means a larger window in which your samples can be detected accurately and precisely. In execution, this means an assay is more sensitive and accurate when detecting lower sample inputs, and on the high end, more tolerant of sample saturation. Figure 2 demonstrates that greater 20 25 30 35 40 Cq -2 -1 0 1 2 Log (qty) Linear fit, IDT probes Linear fit, TaqMan probes Dye 1 20 25 30 35 -2 -1 0 1 2 Cq Log (qty) Linear fit, IDT probes Linear fit, TaqMan probes Dye 2 20 25 30 35 -2 -1 0 1 2 Cq Log (qty) Linear fit, IDT probes Linear fit, TaqMan probes Dye 3 20 25 30 35 -2 -1 0 1 2 Cq log10 concentration Linear fit, IDT probes Linear fit, TaqMan probes Dye 4 20 25 30 35 -2 -1 0 1 2 Cq Linear fit, IDT probes Linear fit, TaqMan probes Dye 5 log10 concentration 20 25 30 35 -2 -1 0 1 2 Cq Linear fit, IDT probes Linear fit, TaqMan probes Dye 6 log10 concentration Figure 2. Dynamic range of TaqMan and IDT probes across four orders of magnitude of template concentration. Reporter dye ABY Cyanine 5 Cyanine 5.5 FAM JUN VIC Delta Cq [(6-plex) - (single-plex)] 6-plex avg. Cq and single-plex avg. Cq -3 -2 -2 -1 -1 0 1 1 20 22 24 26 28 30 32 34 IDT probes TaqMan probes IDT probes TaqMan probes Standard deviation 6-plex avg. Cq 1-plex avg. Cq IDT probes TaqMan probes IDT probes TaqMan probes IDT probes TaqMan probes IDT probes TaqMan probes IDT probes TaqMan probes Figure 3. Mean ΔCq and average Cq values for TaqMan and IDT probes in 6-plex versus singplex assay. TaqMan probes demonstrate smaller differences in performance in 6-plex compared to single-plex assays, as indicated by smaller mean ΔCq values compared to those of IDT probes. or equivalent dynamic range was achieved with TaqMan probes than with IDT probes, for all probes tested in the 6-plex assay. Scaling to a 6-plex assay Multiplexing can be challenging, and probe performance can change when scaled to a higher-plex assay. Deviations in performance can lengthen the design process for multiplexed assays. Figure 3 shows that 67% (4/6) of the time, TaqMan probes have smaller differences in Cq values between 6-plex and single-plex assays than IDT probes. Therefore, TaqMan QSY and QSY2 probes have more consistent and predictable performance when scaling to a 6-plex assay. For Research Use Only. Not for use in diagnostic procedures. © 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. IDT, Iowa Black, Zen, and TAO are trademarks of Integrated DNA Technologies. COL122103 0324 Learn more at thermofisher.com/qsy Figure 4 shows amplification plots for the PCR assays performed with cyanine 5.5–labeled probes. Figure 4. Amplification plots from PCR assays performed with cyanine 5.5–labeled (A) TaqMan probes and (B) IDT probes. Note the similar shapes of the 6-plex and 1-plex amplification curves for the Thermo Fisher product compared to the different shapes of the amplification curves for the IDT product. PCR efficiency PCR efficiency is a specificity indicator for an assay, where 100% represents the exact doubling of template per cycle. Efficiency between 90% and 110% helps ensure more accurate template quantitation and data interpretation. For all 6 probes tested in a 6-plex assay, TaqMan probes had better PCR efficiency than IDT probes. Figure 5 demonstrates that 83% (5/6) of TaqMan probes showed PCR efficiency between 90% and 110% when run in a 6-plex assay, but only 33% (2/6) of IDT probes passed that same metric when run in a 6-plex assay. Data from Figures 1–5 were compiled from two independent sets of 6-plex assays using TaqMan and IDT probes. Double-quenched equivalent IDT probes were utilized where available. Six-plex assays were tested with five concentrations of 10-fold serial dilutions of PCR eciency Dynamic range, orders of magnitude 70% 80% 90% 100% 110% 120% 0 1.0 2.0 3.0 4.0 5.0 Dye 1 Dye 2 Dye 3 Dye 4 Dye 5 Dye 6 Vendor IDT probes TaqMan probes Figure 5. PCR efficiency and dynamic range of TaqMan vs. IDT probes in 6-plex assays. A B the template, plus non-template controls. All probes were tested with the same master mix and analyzed on the Applied Biosystems™ QuantStudio™ 7 Pro PCR instrument. To learn more about multiplexing, visit our multiplex optimization guide. Cyanine 5.5 dye (Thermo Fisher) Cyanine 5.5 dye (IDT)