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December 12, 2023
I. Steitz
Science
Science
qPCR optimization in focus: tips and tricks for excellent analyses
Real-time PCR (qPCR) is undoubtedly an impressive method for measuring the amplification of target DNA in real time. However, some people may despair at the abundance of terms, protocols and kits on the market. This begs the question: are reliable qPCR results only achievable through magic or is it ultimately more about the art of precise fine-tuning?
We can reassure you: in fact, with just a few tricks you can optimize your qPCR so that you can easily achieve reliable and accurate results. Whether you are already an expert in the field of qPCR or just taking your first steps into this fascinating world, this article will guide you through the key factors that will ensure your qPCR assays produce the best possible results. From deciding on the method, to quality control, to optimizing specificity - we have important tips and suggestions for you.
What you should consider when planning your qPCR assay:
High ROX, Low ROX or No ROX?
An internal passive reference dye such as ROX is used in qPCR to normalize the fluorescence signals within the instrument. This is particularly important for older qPCR cyclers that have stationary light sources and detection systems, as different light paths in the device would ultimately result in different fluorescence signals between the wells. A lack of normalization of the measured fluorescence values would lead to distortions and inaccuracies in the qPCR results.
qPCR cyclers from different manufacturers have different optical configurations, which in turn require different ROX concentrations as the base value for normalization. You should therefore clarify with your device supplier whether and if so, which ROX concentration/ROX quantity is required for your device.
In order to fulfil the different requirements, Genaxxon offers appropriately configured qPCR Mastermixes. Our qPCR Mastermixes such as the GreenMasterMix or the ProbeMasterMix are available with different ROX concentrations (No ROX, Low ROX or High ROX). Simply select the appropriate qPCR Mastermix for your specific qPCR cycler.
Probe or SYBR-Green?
There are various methods available for qPCR. The two most commonly used approaches include qPCR with intercalating dyes such as SYBR-Green or Genaxxon's Green DNA dye and qPCR with hydrolysis probes such as TaqMan. In addition, there are other probe-based qPCR methods that do not require hydrolysis, including molecular beacons or scorpion primers. This article is dedicated to the two most common methods - qPCR with intercalating dyes and probe-based qPCR with hydrolysis probes.
Intercalating dyes such as SYBR-Green and Genaxxon's Green DNA dye bind to double-stranded DNA and, after successful binding, generate a fluorescent signal that increases during the PCR reaction in proportion to the amount of PCR products generated. This enables direct detection of amplification in real time. However, careful primer design is crucial to avoid non-specific binding and false positive signals. Therefore, subsequent quality control by means of melting curve analysis is essential when using intercalating dyes.
Hydrolysis probes such as TaqMan, on the other hand, are equipped with a fluorescent reporter dye at the 5' end and a quencher dye at the 3' end. During the PCR reaction, the 5' nuclease activity of the Taq DNA polymerase cleaves the probe, resulting in a fluorescent signal. With each round of amplification, more reporter dye is released, resulting in a specific signal that is proportional to the amount of PCR product.
Hydrolysis probes such as TaqMan are ideal for applications such as SNP genotyping, splice variant analysis and mutation detection by qPCR. In contrast, SYBR-Green based assays are ideal for gene expression or miRNA expression analyses and offer additional benefits, including quality metrics such as melting curve analysis.
But what are the advantages and disadvantages of both methods?
The intercalating dyes such as SYBR-Green and Genaxxon's Green DNA dye are more cost-effective and allow a wider range of applications. However, they require a particularly precise primer design to avoid false signals and subsequent quality control by means of melting curve analysis. On the other hand, probe-based qPCR approaches are more cost-intensive, but also enable multiplexing approaches. They also require less post-processing as no melting curve analysis is performed. Key factors for success in both cases include the selection of suitable primers, optimization of the qPCR master mix and ensuring high-quality, non-fragmented cDNA.
In general, several scientific studies indicate that a properly optimized SYBR-Green assay is as effective as a hydrolysis probe (TaqMan) for gene expression analysis (e.g. Tajadini et. al. 2014: Comparison of SYBR-Green and TaqMan® methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes, Advanced Biomedical Research, 3, 85, DOI: 10.4103/2277-9175.127998).
The choice between the two methods should therefore be based on your specific requirements and available budget. We at Genaxxon offer you specific master mixes for both methods: the ProbeMasterMix for probe-based qPCR and the GreenMasterMix, which already contains the intercalating dye. We also offer special multiplex master mixes such as the 5X qPCR Multiplex PCR Mastermix with an optimised polymerase for robust qPCR performance. Please also read our blog with 3 reasons why you should also use the 5X qPCR Multiplex PCR Mastermix.
Do you now know which ROX concentration your qPCR cycler requires and which qPCR method you want to use? Great, then you should now set up a test assay.
Which components should your test assay contain?
As the qPCR master mixes from different manufacturers have different compositions and the composition of a master mix has a major influence on the PCR, a test assay should be carried out for every new PCR preparation and every time a PCR master mix is changed.
A test assay shows you very quickly whether adjustments need to be made to your protocol. An optimal test assay should include the following components:
- No-template control (NTC) to detect possible cross-contamination. Only water is used instead of a template.
- Template serial dilutions with at least 5 dilution steps to determine the optimal template amount. In addition, you should use genes with known and both low and high expression levels (e.g. housekeeping genes) for testing. This way you can later test which cDNA quantity works optimally for your assay. To minimize the effects of pipetting errors, you should also prepare at least triplicates of your samples.
- Different primer concentrations for testing qPCR specificity to prevent primer dimers.
- NoRT control (noRT) if you use a reverse transcription (RT) from mRNA as a template. As a control, you should use a sample without RT enzyme.
You have made the necessary adjustments to your protocol using the test assay and have now successfully started your qPCR assay? Then you should also carry out a quality validation afterwards.
How to determine the quality of your qPCR assay
Melting curve analysis
In qPCR assays with intercalating dyes such as SYBR-Green or Genaxxon's Green DNA dye, a quality control of the specificity by a melting curve analysis at the end of the qPCR run is essential. This process is carried out by means of continuous temperature increase and simultaneous fluorescence measurement by the qPCR cycler. As the double-stranded DNA products denatures with increasing temperature, a decreasing fluorescence signal is detected. If the change in the slope of this curve is ultimately represented as a function of temperature, a melting curve is obtained.
A specific qPCR result shows only one clear melting peak. The presence of multiple peaks indicates primer dimers or non-specific PCR products, which requires adjustment of the qPCR assay to improve specificity.
Note that the melting temperature itself depends on the length and sequence of the amplicon as well as the salt concentration of the buffer used in the master mix. Changing the master mix can lead to a shift in the melting temperature, but never to a double peak. Multiple peaks always indicate a low specificity of the qPCR.
Ct value
For a comprehensive evaluation of the assay performance, the Ct values play a decisive role, regardless of the method used. In both qPCR methods, the data is visualized in an amplification diagram, with the number of thermal cycles on the x-axis and the detected fluorescence signals on the y-axis.
The Ct value (threshold cycle) of the qPCR is determined on the basis of such a fluorescence curve. The maximum change in the second derivative of the fluorescence curve is determined - i.e. the point at which the fluorescence change no longer increases - and thus exponential amplification is terminated. The cycle in which this is the case is called the "quantification cycle (Cq)" - this corresponds to the Ct value, which is directly dependent on the amount of cDNA - the higher the cDNA input, the lower the Ct value.
Please note that the calculation of the Ct or Cq value can also be performed using other methods. It is important that you always use the same method for your experiment. You should also note that even minor pipetting errors can lead to varying Ct values. It is therefore advisable to analyze at least three replicates of a sample.
How to optimise your qPCR specificity
- Primer concentration: The primer concentration plays a crucial role in the qPCR reaction and should be adjusted accordingly. In general, a lower primer concentration minimizes the risk of primer dimer formation.
- Amplicon length: The length of the amplicon is crucial for the robustness, sensitivity and specificity of a qPCR assay. This applies in particular to probe-based qPCR (e.g. TaqMan assays). Assays with intercalating dyes are less sensitive to amplicon length.
- Annealing temperature: In general, the higher the annealing temperature, the more specific the reaction. Annealing is typically performed at 60°C and the primers used should have a melting temperature > 60°C.
Conclusion
Optimizinnd reliable results and ensure that your qPCR assay is optimized. Do you have further questions or need support with your qPCR? Then feel free to ask us directly: info@genaxxon.com
