qPCR Tips and Tricks

Real-Time PCR Tips & Tricks

Quantitative PCR (qPCR) is a common technique for quantifying nucleic acids in biological and environmental samples. Although many researchers perceive the method to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data. Poorly optimized reactions may lead to misinterpretation or results that are difficult to reproduce.¹

Common challenges in qPCR workflows

Quantitative PCR is an incredibly sensitive way to measure gene expression, yet this sensitivity renders the technique susceptible to bias from small errors or contamination. Pipetting errors are magnified in qPCR workflows, as are discrepancies in primers or reagents. For example, new sets of primers may have different efficiencies than older sets, and old or poor-quality reagents may lead to inconsistences between qPCR assays. Another common problem arises with multiple thermocycler users; researchers using protocols for different targets may complicate or confuse reactions completed in succession if the programs are not checked carefully. When it comes time to publish, researchers may run into difficulties if they have not followed the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, which help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency.²

Overcoming qPCR problems

Although qPCR requires a certain amount of expertise to avoid contamination or replication issues, there are simple steps that a researcher can take to avoid common problems.

One useful workaround is to separate the qPCR workflow into three rooms: one for extraction, one for set-up, and one for qPCR cycling. Pipettes are a primary source of contamination in qPCR workflows. The best way to prevent pipetting mix-ups is to have a set per room, which researchers decontaminate between uses.

Appropriate protective equipment (PPE) helps prevent contamination during liquid handling. Researchers should change gloves between each step of the workflow and use positivedisplacement pipettes with aerosol-resistant filtered tips to prevent aerosol formation near the reactions.

Surface decontamination is also important. 70% ethanol effectively cleans work surfaces before and after setting up and performing a qPCR assay. Periodically cleaning with a 10-15% bleach solutions will also help prevent contamination on benchtops and equipment.

Low retention tubes help ensure better replication of results between runs. These tubes avoid binding nucleic acids, which is especially important when working with dilution series.

Using appropriate buffers for primers is essential, since diluting in water may lead to degradation. Researchers must also consider neutralizing water before using it in a qPCR mix. Diethyl pyrocarbonate (DEPC)-treated water, which is often used in qPCR reactions to inactivate RNase enzymes, can be slightly acidic. Some researchers choose to use pre-prepared qPCR mixes to prevent primer or reagent buffer problems and to accelerate workflows.

When working with a new set of PCR primers, researchers should optimize qPCR reactions by preparing a standard five-point curve with 10-fold dilutions. The PCR efficiency should be at least 90%. Researchers can optimize other qPCR parameters, including annealing temperatures, cycle numbers, and primer design. Part of the optimization process should include determining the correct amount of RNA to use per reaction.

No template controls (NTCs) for each qPCR plate help assess levels of cDNA or gDNA contamination. Controls lacking reverse transcription (NRTs) are also important for assessing levels of gDNA contaminants.¹

Finally, to ensure that experiments meet publication standards, researchers should always follow MIQE guidelines when performing qPCR assays. The guidelines include nine different sections in a checklist, including considerations for performing qPCR and collecting, analyzing, and presenting qPCR data.²

Prevent repeat analysis, increase speed, and reduce cost

Basic prevention procedures go a long way toward optimal and reliable qPCR results, but these qPCR tips will also help scientists save time and money. Analytik Jena’s qTOWER product portfolio significantly reduces cost per sample while providing accurate real-time qPCR results. The thermal cycler contains state-of-the-art heating technology and homogeneity. With its flexible filter module system, researchers can simultaneously analyze 96 to 384 samples without compromising results, and perform up to six-fold multiplexing. The high-performance optics guarantee the outstanding homogeneous excitation and illumination of all individual samples.

References

1. S.C. Taylor et al., “The ultimate qPCR experiment: Producing publication quality, reproducible data the first time,” Trends Biotechnol, 37(7): 761-74, 2019.
2. S.A. Bustin et al., “The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments,” Clin Chem, 55(4):611-22, 2009.