Should I plot starting concentrations or actual copy numbers in 33 nl for each concentration when using Open Array for PCR reactions?

Plotting Strategy for OpenArray PCR Quantification

When using OpenArray for PCR reactions, you should plot the actual copy numbers in the 33 nl reaction volume rather than the starting concentrations to accurately represent the true molecular events occurring in each through-hole reaction. 1

Understanding the OpenArray System and Volume Considerations

The OpenArray technology uses extremely small reaction volumes (33 nl) for PCR amplification, which is significantly different from conventional PCR platforms. This creates important considerations for data representation:

• OpenArray through-holes contain only a tiny fraction of your starting material
• Each 33 nl reaction contains proportionally fewer copies than your starting concentration
• Plotting actual copy numbers in the reaction volume provides more accurate representation of PCR kinetics

Mathematical Conversion for Plotting

To convert from starting concentrations to actual copy numbers in the 33 nl reaction:

1. Calculate the actual copy numbers in each 33 nl reaction:

   • Copy number in reaction = (Starting concentration in copies/ml) × (33 × 10^-6 ml)
   • Example: For 10^6 copies/ml starting concentration:
     • Actual copies in 33 nl = 10^6 copies/ml × (33 × 10^-6 ml) = 33 copies

2. Use these calculated values for your standard curve plotting

Importance of Accurate Volume Representation

Research on digital PCR systems has shown that accurate volume determination is critical for copy number quantification. For example, in droplet digital PCR, an 8% error in assumed reaction volume created significant bias in copy number measurements 2. Similarly, in OpenArray systems, using the actual reaction volume is essential for accurate quantification.

Standard Curve Generation Best Practices

When generating standard curves for OpenArray PCR:

• Plot Ct values against log-transformed actual copy numbers in the 33 nl reaction 1
• Include appropriate quality controls with known low and high copy numbers 1
• Ensure your standard curve has:
  • Slope close to the ideal -3.32 (100% efficiency)
  • R² value >0.99 for high confidence
  • Efficiency between 90-110% 1

Handling Preamplification and Dilution Factors

When preamplification is used:

• Calculate the effective dilution/concentration factor from the preamplification step
• Adjust your final copy number calculations by this factor
• For unknown samples, use the formula:
  • Final copies/ml = (Measured copies in 33 nl) × (Dilution factor) × (1000/33)

Common Pitfalls to Avoid

1. Ignoring reaction volume differences: The 33 nl reaction volume in OpenArray is much smaller than conventional PCR platforms, making direct comparison of starting concentrations misleading.

2. Failing to account for preamplification: Preamplification introduces a multiplication factor that must be incorporated into final calculations.

3. Using inappropriate statistical approaches: For very low copy numbers (<10 copies per reaction), Poisson distribution statistics should be applied as recommended for digital PCR applications 1, 3.

4. Misinterpreting negative results: When reporting undetectable values, always incorporate the sensitivity level of the individual sample to avoid misleading interpretation 1.

By plotting the actual copy numbers present in the 33 nl reaction volume and properly accounting for any preamplification steps, you'll achieve more accurate and reproducible quantification with your OpenArray system.