Complete qRT-PCR Workflow Protocol Step-by-Step

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

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 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

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