Is it desirable to use negative patient samples when performing linearity curves for Quantitative Polymerase Chain Reaction (QPCR)?

Standard Curves for QPCR Should Use Defined Control Materials, Not Negative Patient Samples

For linearity curves in QPCR, you should use well-characterized synthetic standards (RNA, cDNA, or linearized plasmids) rather than negative patient samples. Standard curves must be constructed from serial dilutions of control materials with known, quantifiable target concentrations to establish the log-linear relationship between quantification cycle (Cq) and gene copy number 1.

Why Defined Standards Are Essential

Standard Curve Requirements

• Standard curves require 4-6 ten-fold dilution points of control material with at least three replicates per point to span the dynamic range of the assay 1
• The correlation coefficient (r²) should be ≥0.98 to certify linearity 1
• Acceptable amplification efficiencies range from 90-110%, with ideal efficiency at 100% (slope of -3.32) 1

Problems with Patient Samples

Negative patient samples are fundamentally unsuitable for standard curves because:

• They contain unknown or zero quantities of target sequence, making it impossible to establish the quantitative relationship between Cq and concentration 1
• Patient samples introduce biological matrix effects and potential inhibitors that confound the linearity assessment 2
• The purpose of a standard curve is to create a calibration line from known concentrations—negative samples by definition lack this 1, 3

Appropriate Control Materials

Recommended Standards

Use RNA or DNA standards, with DNA standards offering superior stability 1. The hierarchy of control materials includes:

• Synthetic RNA or cDNA (preferred): These linear materials show lower variability (slope CV 2-7%, efficiency CV 5-8%) compared to plasmids 1
• Linearized plasmids: If using plasmids, linearization is critical to avoid PCR efficiency impacts and bias 1
• Non-linearized plasmids (least preferred): Show significantly higher variability (slope CV 11-15%, efficiency CV 17-19%) and can introduce up to 9.4-fold bias 1

Critical Considerations for RNA Targets

For RT-qPCR assays, RNA standards are preferable because DNA controls bypass the reverse transcription step, failing to account for RT variability 1. This is particularly important when quantifying RNA targets, as the RT step contributes significantly to overall assay variability 1.

Role of Negative Controls

Where Negative Samples Belong

Negative patient samples serve a different purpose entirely:

• Use negative controls to assess contamination background and establish limits of detection, not for standard curves 1
• Include blank extraction controls, blank swab controls, and blank library controls to characterize contamination at different workflow steps 1
• Negative controls help define the cut-off value for positivity (typically 5-10 molecules) 1

Quality Control Framework

Quality control samples at low and high BCR-ABL levels (or equivalent target levels) should be included to monitor assay performance, including maintenance of linearity and stability of standards 1. These QC samples are distinct from the standard curve materials and serve to validate ongoing assay performance.

Common Pitfalls to Avoid

• Never use patient samples of unknown concentration for standard curves—this defeats the quantitative purpose of the assay 1
• Avoid non-linearized plasmids when possible, as they can produce r² values as low as 0.897-0.943, well below the recommended 0.98 threshold 1
• Do not assume vendor-specified titers are accurate—independent quantification of control materials is recommended but reported in only 6% of assays 1
• Report standard curve parameters (slope, efficiency, r², y-intercept) for every run to ensure reproducibility 1