Listed below are some common symptoms in a gene expression/quantification experiment. Select one of the symptoms to view possible causes and solutions.
Related Topics: What Is Real-Time PCR?, How Real-Time PCR Works, PCR Assay Design and Optimization, Allelic Discrimination Experiments, and High Resolution Melt (HRM) Experiments.
Poor signal or no signal
Signal in negative control
Poor reproducibility across replicate samples
Low or high reaction efficiency
Poor signal or no signal in your reaction well(s) may be due to the causes below. Select one of the causes for possible solutions.
Sometimes inhibitors of PCR are carried over from sample preparation (nucleic acid extraction). Common PCR inhibitors include phenol, detergents, proteases, and residual compounds from source materials, such as animal or plant tissue, bodily fluids, or soil preparations. Suboptimal reactions containing inhibitors are highly likely to have poor reproducibility.
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SYBR® Green-based real-time PCR assays require relatively high concentrations of reaction buffer components, such as MgCl2, for efficient amplification.
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Adequate time must be spent at the denaturation, annealing, and extension steps of a thermal cycling protocol to ensure efficient amplification. Insufficient incubation times will not allow complete replication of template, and can result in a lack of amplification signal.
Degraded or sheared nucleic acid template might result in poor amplification signal. Degradation can occur during sample preparation or template storage. Long-term storage of nucleic acid templates in dilute solutions can contribute to template degradation.
Fluorescent hybridization probes may become partially degraded if a probe is subjected to multiple freeze-thaw cycles or long-term storage at temperatures above –20°C. Under these conditions, bonds between the oligonucleotide probe and the conjugated fluorophore become labile. The use of partially degraded or unstable probes results in inefficient fluorophore quenching during real-time PCR, contributing to a high background signal or a gradual increase in baseline fluorescence during thermal cycling.
Signals in negative controls may be due to the causes below. Select one of the causes for possible solutions.
Poor laboratory technique can result in contamination of PCR samples. Contamination will be evidenced by the presence of an amplification signal in negative control samples prepared with all reaction components except the DNA template, referred to as a "no-template" or "primer-only" control.
Primer-dimers may be the result of poor primer design or high primer concentrations.
Example Data: Primer-Dimer Melt Curve
RNA used for reverse transcription (RT)-qPCR must be free of genomic DNA contamination. If the PCR primers for the cDNA template are able to anneal to the genomic copy of the same gene, an amplification signal may be observed in the no-RT controls. If SYBR® Green I is used, perform a melt-curve analysis. Amplified genomic DNA product will have a Tm similar to that of the expected cDNA product.
Example Data: No-RT control samples are represented in black in images below.
Poor reproducibility across replicate samples may be due to the causes below. Select one of the causes for possible solutions.
CT values of replicates can show increased variation due to poor laboratory technique or imprecise pipets.
Some primers are particularly sensitive to thermal cycling conditions, leading to poor reproducibility in amplification reactions.
Low or high reaction efficiency may be due to the causes below. Select one of the causes for possible solutions.
CT values of replicates can show increased variation due to poor laboratory technique or imprecise pipets. This can skew the standard curve, leading to the observation of either low or high reaction efficiency, depending on the type of pipetting error.
In a SYBR® Green assay, the additional fluorescent signal produced by accumulating primer-dimers can skew the standard curve and lead to artificially high reaction efficiencies. Primer-dimers can be the result of poor primer design or high primer concentrations.
Example Data: Primer-Dimer Formation
Fluorescent hybridization probes may become partially degraded when subjected to multiple freeze-thaw cycles or long-term storage at temperatures above –20°C. Under these conditions, bonds between the oligonucleotide probe and the conjugated fluorophore become labile. The use of partially degraded or unstable probes results in inefficient fluorophore quenching during real-time PCR, contributing to extraneous fluorescent signal, which may impact CT values, skew the resulting standard curve, and introduce errors into efficiency calculations.
In some cases, the PCR target may be too long or may contain too much secondary structure to allow efficient amplification by real-time PCR.
Proper assay optimization is essential to the achievement of high-quality results. Nonoptimal assays are highly likely to produce nonspecific products, or insufficient product, resulting in an unexpected shift in PCR amplification efficiency.
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