Curve Shapes

Distinguish real signal from background noise in your qPCR data

“Normal” qPCR amplification curve shape is a sigmoidal shape with what we’ll refer to as three visually distinct apparent phases or regions. The first phase (up to about Cycle 15 or so) is near the baseline with a slow upward trend in the line. The second phase is a strong upward swing in the line, between roughly Cycle 15 and Cycle 30. Finally, the third phase, from about Cycle 30 onward, is a plateau where the amplification signal tapers off and ceases to grow (Figure 1).

Figure 1: Relative fluorescence vs. cycle number. Amplification plots are created when the fluorescent signal from each sample is plotted against cycle number; therefore, amplification plots represent the accumulation of product over the duration of the real-time PCR experiment. The samples used to create the plots in this figure are a dilution series of the target DNA sequence. NTC, no-template control.

The baseline of real-time PCR refers to the signal level during the initial PCR cycles, usually cycles 3 to 15, in which there is little change in fluorescent signal. The low-level signal of the baseline can be equated to the background or the “noise” of the reaction (Figure 2).
The Ct (Depending on your instrument and software, this may alternatively be referred to as CP) is the cycle number at which the fluorescent signal of the reaction crosses the threshold. The Ct is used to calculate the initial DNA copy number, because the Ct value is inversely related to the starting amount of target.
Typically, the threshold is adjusted to the mid-point of the exponential phase of the PCR, at a location suitable for all samples in the experiment. 

Figure 2: The baseline and threshold of real-time PCR 

“Normal” qPCR amplification curve shape is a sigmoidal shape with what we’ll refer to as three visually distinct apparent phases or regions. The first phase (up to about Cycle 15 or so) is near the baseline with a slow upward trend in the line. The second phase is a strong upward swing in the line, between roughly Cycle 15 and Cycle 30. Finally, the third phase, from about Cycle 30 onward, is a plateau where the amplification signal tapers off and ceases to grow (Figure 1).

As the amount of DNA template decreases, the Ct value will increase. If the Ct value gets too high, say a Ct of 35 in a 96 well plate, it can be difficult to distinguish real signal from background.

Sometimes when you are running qPCR experiments, it can be difficult to distinguish real data from background signal, leaving you to wonder whether your data is real.

Useful tips to determine whether you are looking at true signal or background noise:

► Run No Template controls to compare to your experimental samples
 
  • If your sample signal looks similar to your No Template Controls, it’s most likely background.

► Check your replicates:
  • If the Ct value varies among the replicates (ex: 36,37,40), then it is likely not real signal.

► Check on your Ct values:
  • A single copy detection occurs by a Ct of 37 in a 20uL qPCR reaction. Any signal seen later than this is not likely to be real amplification.

► If we change our plot to the linear view:
  • We can see that this curve is actually flat, and no amplification is occurring.

► If we change our plot to the multicomponent view:
  • Shows the curves of each dye in the reaction at each cycle.
  • We can check the curves for exponential amplification, which indicates real signal.
  • While linear upward drift which can be probe degradation at the end of a run.

► Make sure the threshold is going through the exponential phase of the curve.

► Dissociation (Melting) Curves are not performed when using SYBR® Green, the experimental samples should yield a sharp peak at the melting temperature of the amplicon:
  • If the dissociation curve reveals a series of peaks, it indicates that there is not enough discrimination between specific and non-specific reaction products.
  • To obtain meaningful data, optimization of the qRT-PCR would be necessary.

Quiz on possible causes and solutions:

A. Which of the following may indicate a contamination in the reaction?
  1. Exponential amplification in the no template control (NTC)
  2. High noise at the beginning of recorded data, during early cycles
  3. Plateau is much lower than expected
  4. Actual CT is much earlier than anticipated

B. Corrective Steps: Reset Baseline to 3 cycles before the first indication of amplification. Chose the best potential cause and observation that fits to the corrective step:
  1. Exponential amplification in the no template control (NTC) due to contamination
  2. High noise at the beginning of recorded data, during early cycles caused by too many cycles; too much starting material
  3. Plateau is much lower than expected due to a limited reagent; degraded reagents such as the dNTPs or master mix
  4. Jagged signal throughout amplification plot, due to mechanical error; buffer-nucleotide instability; poor amplification or weak probe signal

C. Which of the following may indicate a pipetting error/insufficient mixing of solutions in the reaction?
  1. Technical replicates are not overlapping and have a difference in CT values >0.5 cycles
  2. Jagged signal throughout amplification plot
  3. Actual CT is much earlier than anticipated
  4. Plateau is much lower than expected
  5. High noise at the beginning of recorded data, during early cycles
Answers: A.1 , B.2 , C.1