The difference in signal is due to the master mix composition Reaction was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System with a VIC
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[PDF] Real-time PCR: understanding C - Thermo Fisher Scientific
Real-time PCR, also called quantitative PCR or qPCR, can provide a simple Ct (threshold cycle) is the intersection baseline of master mix B All amplifications were performed using the Applied Biosystems 7500 Real-Time PCR System
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The difference in signal is due to the master mix composition Reaction was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System with a VIC
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Biosystems Quantitative Real-Time PCR 2005 Applied Biosystems ∆Rn Cycles Ct=9 Ct=25 t = 12 h ∆Rn Cycles Ct=9 Ct= What is Multicomponent?
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to allow accurate determination of the threshold cycle (Ct), defined below Applied Biosystems® real-time PCR master mixes contain a red passive reference
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real-time PCR instrument generates an amplification plot OligoPerfect™ designer and Applied Biosystems™ Primer Express™ calculate the initial DNA copy number, because the Ct understand the goal of the assay; specifically, what
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Validation of Target and Control Genes for the Comparative CT Method using real-time PCR technologies developed by Applied Biosystems It assists you in understanding the foundations of relative quantitation and provides guidance for
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Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as Overview Applied Biosystems has developed two types of chemistries used to detect PCR What Is a Quantitation Assay? Ct ( threshold cycle): The fractional cycle number at which the fluorescence passes the fixed
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To understand how real-time PCR works, let's start by examining a sample amount and the CT value obtained during amplification, an optimal qPCR assay is a preformulated kit (TaqMan GMO Soy 35S detection kit, Applied Biosystems )
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APPLICATION NOTE Real-Time PCR: Understanding C
TReal-Time PCR: Understanding C
TFigure 1. A: Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye. In other words, Rn is the reporter signal normalized
to the fluorescence signal of ROX. In this view, Rn is graphed versus cycle. B: ΔRn is Rn minus the baseline, graphed here versus the cycle of PCR. C: Amplification
plot shows the Log (ΔRn) graphed versus cycle. 4.500 4.000 3.500 3.000 25002000
1500
1000
0500
Rn
Cycle Number
3.500 3.000 2.500 2.000 15001000
0500
0000 -0500
ΔRn
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Cycle Number
1000e + 001
1000e + 000
1000e - 001
1000e - 002
1000e - 003
1000e - 004
ΔRn
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Cycle Number
Exponential phase
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Threshold
Threshold
Baseline
Introduction
Real-time PCR, also called quantitative
PCR or qPCR, can provide a simple
and elegant method for determining the amount of a target sequence or gene that is present in a sample. Its very simplicity can sometimes lead to problems of overlooking some of the critical factors that make it work. This review will highlight these factors that must be considered when setting up and evaluating a real-time PCR reaction.Factors that can Influence C
T C T (threshold cycle) is the intersection between an amplification curve and a threshold line (Figure 1B). It is a relative measure of the concentration of target in the PCR reaction. Many factors impact the absolute value of C T besides the concentration of the target. We will discuss the most common template- independent factors that can influence C T and describe how to evaluate the performance of a real-time PCR reaction.Figure 1 shows several parameters
of the real-time reaction amplification plot. The exponential phase in Figure1B corresponds to the linear phase in
Figure 1C. The threshold must be set
in the linear phase of the amplification plot in Figure 1C. The C T value increases with a decreasing amount of template.However, anything from the reaction
mix or instrument that changes the fluorescent measurements associated with the C T calculation will result in template-independent changes to the C T value. Therefore, the C T values from PCR reactions run under different conditions or with different reagents cannot be compared directly.The Effect of Master Mix Components
The fluorescent emission of any
molecule is dependent on environmental factors such as the pH of a solution and salt concentration. Figure 2 shows the raw fluorescence data of aTaqMan
probe in the background of two different master mixes. Note that the fluorescence intensity is higher inMaster Mix A even though the target,
probe and ROX concentrations are the same in both cases.0510 15 202530
BinAmplitude
6.00 E + 3
5.00 E + 3
4.00 E + 3
3.00 E + 3
2.00 E + 3
1.00 E + 3
0.00 VIC ROXMaster Mix A
Master Mix B
Figure 2. Raw fluorescence data obtained with one
assay and two master mixes with the same ROX level. The difference in signal is due to the master mix composition. Reaction was performed on anApplied Biosystems 7900HT Fast Real-Time PCR
System with a VIC
MGB probe. The X axis shows
the emission wavelength of the fluorophore and the Y axis shows the intensity of the emission.The resulting ΔRn value will, therefore,
vary as shown in Figure 3. Note that the baseline fluorescence signals, in a template-independent factor, are different for the two master mixes (Figure 3A). Variations in C T value do not reflect the overall performance of the reaction system (Figure 3B). Master mixes with equivalent sensitivity may have different absolute C T values. ROXPassive Reference Dye
The Rn value is calculated as the ratio
of FAM fluorescence divided by theROX fluorescence. Therefore, a lower
amount of ROX would produce a higherEfficiency of a PCR Reaction
The efficiency of a PCR reaction can
also affect C T . A dilution series amplified under low efficiency conditions could yield a standard curve with a different slope than one amplified under high efficiency conditions. In Figure 5, two samples (X and Y) amplified under low and high efficiency conditions show different C T values for the same target concentration. In this example, although the high efficiency condition (the blue curve in Figure 5) gives a later C T at high concentration, it gives better sensitivity at low target concentration.Rn value assuming FAM fluorescence
signal stays the same. This would lead to an increase in baseline Rn and subsequently a smallerRn as well as a
different C T value. The different C T value obtained by lowering the ROX level has no bearing on the true sensitivity of the reaction, but can have other unintended consequences. Low ROX concentration can result in increased standard deviation of the C T value, as shown in Figure 4. The greater the standard deviation, the lower the confidence in distinguishing between small differences in target concentration (see the precision section on the next page).1 2 3 3 3 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
0.866 0.766 0.666 0.566 0.466 0.366 RnCycle Number
AMaster Mix A
Baseline A
Baseline B
Master Mix B
1 2 3 3 3 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
1.000 0.100 0.010 0.001ΔRn
Cycle Number
BMaster Mix B
Master Mix A
CTB CTAFigure 3. Master Mix A and Master Mix B were used to amplify RNase P in equal amounts of human gDNA using the Applied Biosystems 7500 Real-Time PCR
System. Figure 3A shows the Rn versus cycle number and the baselines for both reactions. Figure 3B shows the Log (ΔRn) versus cycle number. The threshold
(green) is set at the same level for both master mixes. The C T value of Master Mix B (C T