qPCR Principles, Experimental Workflow and Results Analysis

critical

No amplification — Ct value missing or > 40

Amplification plot is flat for both target and reference; software reports "no Ct" or Ct > 40 even for positive controls.

💡 5 · ✓ 5

critical

False Positive PCR Results from DNA Contamination

No-template controls (NTC) or negative controls show unexpected amplification. Particularly problematic in sensitive applications like human identification qPCR where trace DNA contamination produces false positives.

💡 3 · ✓ 3

severe

Low Fluorescence Due to Inadequate Probe Labeling or Quenching

Low or absent fluorescence in both test sample and positive control; correct PCR product is visible on gel; one probe in multiplex shows consistently high background with no amplification signal; background fluorescence equivalent to water control

💡 5 · ✓ 6

severe

Reference gene is unstable — normalization fails

Reference gene Ct varies > 1 cycle between samples; trend of reference vs target gene disagrees; normalization gives strange results.

💡 4 · ✓ 4

severe

Plate or Tube Melting and Adhering to Block

After PCR program completion, plates or tubes are found melted or stuck to the thermal cycler block, making removal difficult and potentially damaging samples. Plastic shows signs of thermal degradation.

💡 3 · ✓ 3

severe

PCR Sample Evaporation and Loss During Cycling

Visible reduction in reaction volume after thermal cycling, with condensation on tube caps or film. May result in concentrated reagents, failed reactions, or inability to recover product.

💡 3 · ✓ 3

severe

PCR ReadyMix Works for PCR but Fails in qPCR

ReadyMix produces amplification in standard PCR but completely fails in real-time qPCR applications; equivalent products from other suppliers work well

💡 3 · ✓ 3

severe

PCR Efficiency Greater Than 120% with Inconsistent ΔCq

PCR efficiency calculated from standard curve is greater than 120%; ΔCq between 10-fold dilutions is much less than expected 3.3 cycles (e.g., 1.5 cycles); standard curve gradient indicates abnormally high efficiency

💡 4 · ✓ 6

severe

Low PCR Product Yield

Desired PCR fragment appears faint or barely visible on gel electrophoresis

💡 14 · ✓ 14

moderate

Atypical amplification curves (not the canonical S-shape)

Curves cross threshold but are not the canonical sigmoidal shape — may have weird plateaus, early plateaus, or noisy baselines.

💡 5 · ✓ 5

moderate

Nonspecific Amplification or Multiple Melt Peaks in SYBR Green qPCR

Multiple products or melt curve peaks appear in SYBR Green qPCR after ChIP, indicating amplification of off-target sequences. Gel electrophoresis may reveal bands at incorrect sizes or multiple bands.

💡 4 · ✓ 5

moderate

Variable qPCR Data Across Wells

Quantitative PCR shows inconsistent Cq values or fluorescence intensities between technical replicates in different wells, despite identical reaction setup. Well-to-well variability exceeds acceptable coefficient of variation.

💡 2 · ✓ 2

moderate

PCR Tube Crushing or Deformation Under Lid Pressure

PCR tubes become crushed, collapsed, or deformed after thermal cycling, potentially causing sample loss or compromised seal integrity. Tubes may show visible damage especially when using tube strips.

💡 3 · ✓ 3

moderate

PCR Tube Caps Popping Off During Cycling

Individual tube caps become dislodged during thermal cycling, exposing samples to evaporation and potential contamination. Caps are found loose or completely separated from tubes after run completion.

💡 3 · ✓ 3

moderate

Abnormal Amplification Plots Due to Excessive Template Concentration

Very low Cq values for concentrated samples; amplification plots are not regularly spaced and appear abnormal; background fluorescence is significantly higher; minimal fluorescence yield through the reaction

💡 3 · ✓ 4

moderate

Amplification Plots Dip Below Zero Due to Incorrect Baseline Settings

Amplification plots are clearly abnormal with sections dipping below zero dR; data cannot be used as presented; plots appear distorted

💡 3 · ✓ 4

moderate

Incorrect Reaction Efficiency Due to Oligo Binding to Non-Molecular Biology Tubes

Variable and incorrect standard curve efficiency when using serial dilutions; effect more pronounced when same dilution series stored at 4°C and reused; inconsistent differences between amplification plots; problem resolves when different operator uses different tubes

💡 4 · ✓ 5

moderate

Low qPCR Fluorescence Signal from Optical Issues

Quantitative PCR shows weak fluorescence signal across all wells, making accurate quantification difficult. Signal intensity is lower than expected despite adequate template and reagent quality.

💡 2 · ✓ 2

moderate

Pressure-Sensitive Film Not Adhering to Plate

Sealing film peels away from plate wells either before or during thermal cycling, leading to sample evaporation or cross-contamination. Film appears to have poor bonding with well rim surfaces.

💡 2 · ✓ 2

moderate

Irregular Standard Curve Spacing Due to Sample Inhibitors

Cq data for standard curve dilutions are irregularly spaced; ΔCq between dilutions is inconsistent and decreases with increasing dilutions; replicates are precise but pattern is abnormal

💡 3 · ✓ 5