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

Complete Absence of PCR Product

No bands visible on agarose gel after PCR amplification, indicating complete reaction failure.

💡 6 · ✓ 6

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

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

High Background Signal Due to Insufficient ChIP Washing

Elevated background signal appears across all samples including negative controls, with nonspecific amplification in qPCR. PCR products may show multiple bands or high Ct values in control regions.

💡 4 · ✓ 5

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

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

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