If you've ever scratched your head over inconsistent qPCR results or unexpected variation in NGS workflows - especially when identical samples yield different Ct values - you're not alone. One of the most overlooked culprits? The plates themselves.
Most PCR and qPCR experiments rely on one-component plates made entirely of polypropylene (PP). Polypropylene is widely used because it’s chemically inert and allows for ultra-thin tube walls, which help with fast thermal cycling. But PP has a critical weakness: it starts to soften at PCR-relevant temperatures and expands significantly when heated. This thermal expansion can break the seal between the plate and film or foil, especially at the edges, leading to evaporation, and ultimately, skewed results.
The Evaporation Effect: Why It Matters
During PCR, evaporation is no small matter. It can lead to:
- Changes in concentration and pH of your reaction mix
- Reduced enzyme efficiency
- Significant variation in Ct values, particularly in the outer and corner wells
In fact, data from Azenta Life Sciences shows that 60% of reported evaporation-related issues in 96-well plates occur in the outer wells.
Enter Two-Component Plates: A Smarter Design
Two-component plates, such as FrameStar™ from Azenta Life Sciences, pair the best of both materials:
- Tube wells made of polypropylene, for optimal thermal transfer
- A rigid polycarbonate frame, which resists heat-induced distortion
Polycarbonate has a Vicat Softening Temperature above 145°C, far higher than the ~90°C of polypropylene. That means while the inner tubes still do their job, the frame stays flat and stable, keeping seals tight and evaporation at bay.
Real-World Impact: Less Distortion, More Reliable Data
In controlled experiments, one-component plates showed up to 2mm of thermal expansion after PCR — enough to warp the plate and loosen seals. In contrast, two-component FrameStar plates showed negligible distortion (just 0.03mm).
The effect on evaporation is dramatic:
- One-component plates lost up to 28% of volume in outer wells
- Two-component plates kept that loss under 6%
Better Sealing Options Mean Even Better Results
Seal integrity matters just as much as plate design. Heat seals outperform adhesive seals by maintaining better contact, even under pressure and temperature changes. Heat sealing offers even greater protection against evaporation compared to adhesive seals. Combine heat sealing with a two-component plate, and the difference is clear:
- With 500nl reactions, only 82% of wells retained detectable liquid in standard plates using adhesive seals, versus 97% in two-component plates with heat seals
- At higher volumes, two-component plates maintained 100% retention
Final Thoughts: Precision Starts with the Right Plate
Small differences in your consumables can make a big difference in your results. Whether you're running a few reactions or scaling up for high-throughput screening, the design of your PCR plate impacts the quality of your data.
Two-component PCR plates offer a robust, reliable alternative to traditional all-PP designs - reducing evaporation, improving consistency and enabling lower reaction volumes. This can save you meaningful time, reagents, and money. So next time your qPCR results don’t quite add up, take a closer look at your plates. Switching to a two-component plate could be the upgrade your data has been waiting for.
Tech Note: Influence of PCR Consumables on the Accuracy of Real-Time PCR Experiments | Azenta Life Sciences