The 10 Most Common PCR Mistakes and How to Avoid Them

PCR (Polymerase Chain Reaction) is a cornerstone of molecular biology, yet even experienced researchers can encounter pitfalls that compromise results. Whether you are performing conventional PCR or real-time qRT-PCR, avoiding common errors is essential for reproducibility and accuracy. Here, we outline the 10 most frequent PCR mistakes and provide tips to optimize your experiments.

1. Poor Primer and Probe Design

Effective PCR starts with proper primer and probe selection. Using primer design software ensures optimal parameters, including melting temperature (Tm), primer/probe complementarity, secondary structure, and amplicon size. Avoid long runs of identical nucleotides, and for eukaryotic targets, design primers spanning exon-exon junctions to prevent genomic DNA amplification. Proper design maximizes specificity and PCR efficiency.

2. Using Low-Quality RNA

Degraded or impure RNA reduces reverse transcription efficiency and PCR yield. Use fresh tissue or RNA stabilized with preservation solutions. For qRT-PCR, amplicons are typically 70–250 bp, allowing some RNA degradation, but for quantitative accuracy, full-length RNA is ideal. If RNA is partially degraded, design primers in internal gene regions.

3. Not Using Master Mixes

A master mix combines all reaction components, reducing variability across multiple PCR reactions. Using master mixes improves reproducibility and minimizes pipetting errors. Adding reference dyes such as ROX further standardizes results.

4. Introducing Cross-Contamination

PCR is highly sensitive; even trace DNA contamination can produce false positives. Routinely clean all surfaces with DNA-degrading solutions (DNAzap) and always include a No Template Control (NTC). If the NTC shows amplification, contamination is present.

5. Not Using a “–RT” Control

Genomic DNA contamination is almost inevitable in RNA samples. Include a minus-reverse transcriptase control (NAC) to confirm RNA-specific amplification. Amplification in the NAC indicates contaminating DNA, which could skew qRT-PCR results.

6. Using an Inappropriate Normalization Control

Normalization is critical for accurate gene expression analysis. Choose a stable endogenous control, such as 18S rRNA, instead of variable housekeeping genes like GAPDH or β-actin. This ensures consistent correction for sample-to-sample variation.

7. Skipping Dissociation (Melting) Curves with SYBRGreen

When using SYBRGreen, always perform melting curve analysis to verify specificity. A single sharp peak indicates a specific product, while multiple peaks suggest non-specific amplification. Proper optimization ensures that fluorescence accurately reflects your target accumulation.

8. Incorrect Baseline and Threshold Settings

Accurate Cq (quantification cycle) values require correctly set baselines and thresholds. Set the baseline two cycles before the most abundant sample and choose thresholds during the exponential amplification phase. Misadjustment can lead to erroneous quantification.

9. Poor PCR Efficiency

Reaction efficiency should range 90–110%, corresponding to a slope of 3.1–3.6. Factors like amplicon length, secondary structures, and primer design affect efficiency. Optimize primers or select alternative amplicons to improve reaction performance.

10. Using an Inappropriate Range for Standard Curves

Standard curves validate reaction efficiency and enable RNA quantification. Ensure the curve spans above and below the expected target abundance. Including RNA amounts near detection limits helps distinguish specific from non-specific products.

Conclusion

Avoiding these common PCR mistakes can dramatically improve the reliability of your experiments. From primer design and RNA quality to controls, efficiency, and data analysis, following best practices ensures that your qRT-PCR results are accurate and reproducible.