Successful qPCR assays require specific and efficient application of products. Both the target sequence and primers affect amplification specificity and efficiency, thus resulting in the accuracy of a qPCR assay.

Therefore, it is important to be careful when designing primers and choosing target sequences. It is recommended that PCR primers specifically validated and designed for qPCR probes and assays with the target of interest be used.

What a Probe-Based qPCR is

It is a PCR technique that uses fluorescently labelled sequences specific to DNA oligonucleotides called probes to obtain specific and accurate results.

Using a probe requires us to design primers, as well as probes that will make experiments more costly and time-consuming than using a dye-based qPCR option.

How It Works

To start designing probe-based qPCR, we will require a fluorescent reporter dye on just one end of our probes and the right quenching element on the other side to ensure fluorescent doesn’t absorb the light the reporter emits.

The quencher and fluorescent reporter dye are located near each other to ensure the quencher prevents fluorescence. In addition, it is crucial to consider the probes’ and primers’ locations when designing them.

Probes must be between primers in the sequence. During PCR design, probes bind and are located downstream of primers. Afterward, the quencher and fluorescent reporter dye are separated by what is called Taq DNA polymerase, which cleaves probes.

Basic Lab Techniques for a qPCR Assay

The right lab technique is crucial yet an overlooked step required for the success of PCR design. For good results, you need to minimize cross-examination between different samples and avoid carryover of the nucleic acid from the first experiment to the second one. The following precautions may help you to prevent contamination issues:

• Use a screw-capped tube for reaction setup and dilutions.
• Frequently change gloves during reaction setup and sample preparation.
• Wide down your workstation with a dilute solution of bleach.
• Use an aerosol-barrier pipet tip or shaftguard pipet.
• Prepare samples in designed clean rooms, benchtop workstations, or hoods equipped with UV lamps.
• Use a PCR-grade reagent and water dedicated for PCR uses only.

Designing Tips

If you are looking to design a qPCR probe for gene quantification, you will need tips from the pros. Some of these tips include the following:

• Avoid Cross-Contamination

Every surface in PCR areas must be decontaminated routinely to avoid cross-contamination. You may use DNA decontamination solutions that destroy DNA.

• Prioritize Quality RNA

We should either prepare RNA from refreshed tissues or those treated with RNA stabilization solutions, like Invitrogen™ RNAlater® Stabilization Solution. This way, some RNA degradation will be tolerated. If it won’t be possible to completely use intact RNA, consider designing primers to anneal to internal regions of the gene of interest.

• Use Master Mix

A Master Mix containing the required reagents for qPCR will help reduce well-to-well and sample-to-sample variation and may improve reproducibility. To take this variation even further, you can use a master mix containing a reference dye, like ROX.

The bottom line is that probe-based qPCR is a great tool for quantification and detection of nucleic acid. However, considering the impact that sample contaminants have on primer annealing and polymerase efficiency, we must follow a stepwise and rigorous procedure to verify samples, validate primers, and plan experiments.