Gene Expression/Quantification Experiments

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.

SYBR^® Green-based real-time PCR assays require relatively high concentrations of reaction buffer components, such as MgCl₂, for efficient amplification.

• MgCl₂ concentration: 3.0–6.0 mM
• dNTP concentration: 200–600 μM each dNTP – increasing the [dNTP] will require an increase in [MgCl₂]
• Concentration of Taq DNA polymerase: 1.25–4.5 U/50 μl reaction
• Primer concentration: 100–500 nM; an asymmetric primer concentration may be helpful
• Fluorescent probe or intercalation dye concentration: 200–300 nM

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.

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.

• Wear gloves
• Use screwcap tubes
• Use aerosol-resistant filter tips
• Use calibrated pipets dedicated to PCR
• Use PCR-grade water and use it only for PCR
• Use a no-template control to verify absence of contamination
• Prepare a master mix with sufficient volume to prepare all replicate samples

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.

C_T values of replicates can show increased variation due to poor laboratory technique or imprecise pipets.

• Wear gloves
• Use screwcap tubes
• Use aerosol-resistant filter tips
• Use calibrated pipets dedicated to PCR
• Use PCR-grade water and use it only for PCR
• Use a no-template control to verify absence of contamination
• Prepare a master mix with sufficient volume to prepare all replicate samples
• Add template to master mix before pipetting into reaction vessels
• Avoid pipetting less than 5 μl

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.

Some primers are particularly sensitive to thermal cycling conditions, leading to poor reproducibility in amplification reactions.

C_T 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.

• Wear gloves
• Use screwcap tubes
• Use aerosol-resistant filter tips
• Use calibrated pipets dedicated to PCR
• Use PCR-grade water and use it only for PCR
• Use a no-template control to verify absence of contamination
• Prepare a master mix with sufficient volume to prepare all replicate samples
• Add template to master mix before pipetting into reaction vessels
• Avoid pipetting less than 5 μl

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

Example Data: Primer-Dimer Melt Curve

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 C_T 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.

• Amplify a template region of 75–150 bp
• Avoid secondary structure if possible
• Use an annealing temperature above the melting temperature (T_m) of any template secondary structure
• Avoid templates with long (>4) repeats of single bases
• Maintain a GC content of 50–60%
• Analyze secondary structure using the DNA mfold server created by Dr Michael Zuker (http://www.bioinfo.rpi.edu/applications/mfold) or an equivalent primer-design program

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.

• MgCl₂ concentration: 3.0–6.0 mM
• dNTP concentration: 200–600 μM each dNTP — Increasing the [dNTP] will require an increase in [MgCl₂]
• Concentration of Taq DNA polymerase: 1.25–4.5 U/50 μl reaction
• Primer concentration: 100–500 nM; an asymmetric primer concentration may be helpful
• Fluorescent probe concentration: 200–300 nM