Good qPCR amplification curves should look like Figure 1. During probe-based qPCR assays, fluorescence is released from the probe as DNA polymerase copies the template or target nucleic acid sequence. The exonuclease activity of thermostable DNA polymerase hydrolyzes the probe, thus separating its 5' fluorophore from the quencher. The fluorescence of the degraded probe is divided by the fluorescence of an internal reference dye, ROX, to find the normalized fluorescence value, Rn. This value is then plotted for each cycle (Figure 1A).

Once the fluorescence values increase enough to reach the detection threshold of the instrument, the fluorescence will continue to accumulate for each cycle until the amount of the amplicon competes with the primers for hybridization, and a plateau in fluorescence accumulation is reached.

In Figure 1B, the intersection of the amplification curve and the threshold is defined as the Cq value, an approximate measure of the concentration of target sequences in the sample. The Cq values are evaluated relative to an internal control gene or to a standard curve created with serial dilutions of a known amount of target sample.

Ideal or expected qPCR amplification curve data

qPCR is a complex, multifaceted process, and several factors can cause suboptimal amplification. So sometimes your curves look like these (Figures 2 and 3):

Suboptimal qPCR amplification curves
Figure 2. There is strong preference for using the non-targeted strand sequence as the HDR donor sequence for Cas12a HDR. Jurkat cells were transfected by electroporation with Alt-R A.s. Cas12a Ultra RNPs targeting multiple loci along with Ultramer HDR donor oligos containing a 6-base EcoRI insert and 40-nucleotide homology arms. These single-stranded oligodeoxynucleotides (ssODNs) contained either the targeted or non-targeted strand sequence and were directly compared with each other. Editing efficiency and HDR were measured by NGS. The targeted strand donors had reduced total editing and overall HDR rates, most likely due to interference with Cas12a binding to the desired DNA sequence. In sites where editing was equal, the strong preference for non-targeted sequence was not observed. We recommend always using the non-targeted strand sequence as the donor oligo sequence for HDR when using Cas12a.
Suboptimal qPCR amplification curves
Figure 3. For Cas12a HDR, walking the insert 2–6 bases from the cut site toward the PAM can dramatically increase HDR efficiency. HEK-293 cells were transfected by electroporation with wild-type A.s. Cas12a RNPs targeting human HPRT along with Ultramer HDR donor oligos containing a 6-base EcoRI insert and 40-nucleotide homology arms. Editing efficiency was measured by T7EI assay. HDR was measured by EcoRI cleavage of target amplicons; error bars represent the standard deviation of 3 technical replicates. Positioning the insert (the EcoRI site) 2–10 bases closer to the PAM increases the rate of HDR by fully disrupting the protospacer sequence, while positioning the insert further away from the cut site is disfavored. Optimizing the insert site location greatly improves the rate of HDR.

You might observe something very different from what you expect (Figures 4-7):

qPCR amplification curves of low height Amplification of the No-Template Control (NTC) qPCR dilution series produce inconsistent Cq intervals Amplification curves with delayed Cq values

Thus, troubleshooting or generation of additional data may be required to achieve optimal qPCR results. But first you need to determine what is causing the flawed data.

The Real-time qPCR guide: Part 3—troubleshooting uses stylized drawings like the above and real amplification plots to help you identify the factors that could be compromising your results. These might include: sample degradation, low target copy number, incorrectly assigned dye detector, or overlapping fluorescent emission spectra. Just match your data to one of the images that best represents your own data and refer to the indicated guide section to learn what can cause such amplification curves and how to improve them. Click here to register to download the Real-time qPCR guide: Part 3—troubleshooting. 

For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. Doc ID: RUO23-1689_001