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

High Signal in All Samples Including No Template Control

PCR amplification occurs in all samples including no template controls (NTC), producing Ct values or visible bands in negative controls. This indicates systemic contamination rather than specific ChIP enrichment.

💡 4 · ✓ 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

Poor Ct reproducibility — high within-group variability

Technical replicates of the same sample have SD > 0.5 cycles; standard curve linearity is poor; loading order influences results.

💡 5 · ✓ 5

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

No or Low PCR Amplification Due to Plastic Consumable Issues

PCR reaction fails to produce expected amplification or yields significantly reduced product, despite optimized reagents and cycling parameters. The issue traces to suboptimal thermal transfer or contamination from the plastic vessel itself.

💡 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 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

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

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

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

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