SYBR Green is a tried-and-true reaction chemistry for quantitative real-time PCR (qPCR). It is considered simple, cost-effective and accurate. It’s not always as straightforward a method as you might think, however, and a little attention to optimizing it goes a long way to help ensure data quality. Correctly designing and optimizing primers and incorporating a target-specificity quality control step will help ensure you can trust your data.
How does SYBR Green work?
SYBR Green is a free-floating fluorescent dye that binds to double-stranded DNA and increases in fluorescence when bound. In a qPCR reaction, as the primers and DNA polymerase duplicate the template strand, SYBR Green binds to double-stranded DNA as it forms. The qPCR instrument measures this fluorescence, which enables quantification of the DNA present in the original sample.
The advantage of SYBR Green is that, because it binds to all double-stranded DNA, it does not require specific probes, which can help reduce cost. It is a one-size-fits-most method ideal for researchers performing gene expression analysis experiments, but this advantage comes with some drawbacks. Binding to all double-stranded DNA means that SYBR Green cannot multiplex—that is, it cannot analyze multiple, specific targets in the same reaction simultaneously—and it also fluoresces in response to primer dimers and non-specific amplification. Additionally, different SYBR Green assays provide different amounts of leeway regarding primer concentrations in the master mix. Optimizing a SYBR Green experiment means finding ways to reduce all this signal noise and making sure that the fluorescence reading corresponds to amplification of the specific target. The steps required to achieve this certainty start during the assay design and optimization phase.
How good is your cDNA, really?
A key step in studying gene expression with qPCR is the synthesis of cDNA. This is DNA reverse transcribed from an RNA template—in this case, the mRNA that is your actual target for study. The enzyme used to create cDNA, reverse transcriptase, can introduce bias where the amount of cDNA is not consistently proportional to the amount of RNA in samples. This can be investigated by performing qPCR to amplify the target of interest and an endogenous control from a dilution series of cDNA samples of known concentrations, generating standard curves. By comparing the qPCR standard curves of the target assay and the endogenous control assay, you can determine which RNA quantities introduce RT bias. For more information on this, check out our Taq Talk video on optimizing SYBR Green experiments.
SYBR Green dye non-specifically binds to double-stranded DNA, so the best way to check if a SYBR Green qPCR reaction amplified a single target without primer dimer formation is with melt-curve analysis. Melt-curve analysis takes place after a qPCR run and serves to verify that the fluorescence detected during the run come from a single amplicon. The nonspecific nature of SYBR Green as a fluorophore makes melt-curve analysis one of the very few ways to confirm that target-specific amplification occurred. If some of the fluorescence generated is due to primer dimers or non-specific amplification, your relative quantification of target is suspect, so when starting up a new SYBR green assay, always perform melt-curve analysis as a QC step to help confirm target specificity.
During melt-curve analysis, the temperature is steadily increased from about 60°C to about 95°C, denaturing the DNA within. In a qPCR reaction chamber with a single amplicon, there is a smooth reduction in the fluorescence throughout as the DNA within denatures, until the last of it is denatured and the remaining fluorescence drops precipitously to background. The temperature at the halfway point of the drop-off is the presumed melting temperature of that PCR product. Choose the derivative melt curve view in your analysis software to convert the drop-off to a peak.
Observing a single peak suggests, but does not prove, that a single product was amplified. This can be confirmed by analyzing the PCR amplification product by agarose gel electrophoresis. Observing a single band on the gel is further evidence that a single product was amplified.
Observing multiple peaks, shoulders on the main peak, unusually wide peaks or asymmetrical peaks suggests that primer-dimers formed or that non-specific amplification or some other anomaly occurred.
Two main sources of multiple peaks include non-specific amplification and primer-dimer formation.
- Non-specific replication is when the primers bind to non-target sequences and facilitate amplification. Increasing primer specificity can help prevent this non-specific amplification, causing the primers to bind only to the desired target.
- Primer-dimer formation occurs when primers bind to other primers. This can happen if primers are symmetrical enough to interact with themselves or with their own reverse primers. Sometimes, reducing primer concentration in the reaction or increasing the annealing temperature is sufficient to deter primer-dimer formation, but redesigning primers to make this less likely is sometimes necessary.
Some curve shapes, including asymmetrical or very wide peaks, indicate a more unusual problem that is more difficult to diagnose. Especially strange-looking melt-curve peaks may merit re-running or redesigning the experiment or running instrument diagnostics. For more information on melt-curve analysis, enjoy our Taq Talk episode on how to perform this important quality-control step and read our white paper about how to optimize a SYBR Green qPCR assay.
Understanding how SYBR Green assays work and designing experiments and protocols that address their basic characteristics helps ensure every experiment you run is as successful as possible. Always test new SYBR Green assays with melt-curve analysis QC to help confirm target specificity so you can trust the accuracy of your data.
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