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

Insensitive Assay with Abnormal Amplification Due to Probe Secondary Structure

Assay is insensitive and amplification plots look abnormal with pronounced drift of the baseline; fluorescence signals are weak or irregular

💡 3 · ✓ 4

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

Abnormal melt curve (multiple peaks or shoulder)

Melt curve shows multiple peaks or a shoulder, Tm does not match expectation, gel shows multiple bands.

💡 5 · ✓ 5

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

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

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

Primer Dimer Formation at Low Template Concentrations

Low concentration data points do not fit linear standard curve profile; NTC shows amplification with lower Tm and broader melt peak than positive samples; primer dimers visible on gel, inversely proportional to template concentration

💡 4 · ✓ 5

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