No. | Issue Description | Potential Causes | Recommended Solutions |
1 | Poor Standard Curve Gradient | Inaccurate pipetting or dispensing | Verify pipette and tip integrity |
Incomplete plate washing | Ensure proper wash cycles and buffer volume per well | ||
2 | Weak or No Color Development | Insufficient incubation time | Adhere to recommended incubation duration |
Incorrect temperature | Maintain specified incubation temperature | ||
Inadequate reagent volume or missed steps | Confirm accurate reagent dispensing in correct sequence | ||
3 | Low OD Values | Improper microplate reader settings | Verify wavelength and filter configuration; pre-warm the reader |
Excessive or inadequate washing (e.g., prolonged buffer retention) | Follow manufacturer's washing protocol strictly | ||
4 | High Coefficient of Variation (CV) | Pipetting errors | Retrospectively review or validate pipetting accuracy and consistency |
Uncalibrated pipettes | Perform regular pipette calibration and testing | ||
Residual liquid or fingerprints on plate bottom | Inspect wells for contaminants; ensure bubble-free dispensing | ||
5 | Elevated Background | Incomplete plate washing | Optimize wash steps per protocol; check automated washer nozzles for clogs |
Contaminated shared reagents (e.g., ultrapure water) | Replace with fresh, uncontaminated reagents | ||
Contaminated equipment (e.g., pipettes, centrifuges) | Dedicate pipettes for assays; use sterile filtered tips | ||
Cross-contamination from lab workflows (e.g., cell culture) | Segregate ELISA workspaces | ||
Incorrect reagent dilution (e.g., wash buffer, detection antibody) | Re-prepare at specified dilutions | ||
6 | Discrepancy vs. Expected Performance | Improper kit storage | Store reagents as instructed |
Expired reagents | Verify all kit components are within expiration dates | ||
Protocol deviations | Train personnel rigorously; enforce adherence to critical steps (e.g., concentrations, incubation times, volumes) |
No. | Issue Description | Potential Causes | Corresponding Solutions |
1 | No CT Value | Incorrect PCR program setup; improper fluorescence signal detection steps | Verify fluorescence selection and acquisition settings in the program |
Primer/probe degradation | Assess potential degradation via PAGE electrophoresis | ||
Template degradation or insufficient loading volume | If degradation occurs, consider sample impurity introduction or freeze-thaw cycles | ||
2 | Values Outside Standard Range | Errors in reaction mixture preparation calculations | Double-check calculations during master mix preparation |
3 | Suboptimal Standard Curve | Reference DNA dilution, mixing, or pipetting inaccuracies leading to non-gradational references | Ensure pipetting precision, avoid liquid adherence, homogenize thoroughly (check ROX signal), and maintain appropriate dilution gradients |
Reference material degradation | Limit freeze-thaw cycles to specified counts | ||
Presence of inhibitors in the template | Check ROX signal; re-dilute template | ||
4 | Delayed CT Value | Degradation of PCR components or insufficient loading volume | Verify ROX signal; validate component integrity via gel electrophoresis; reduce dilution and repeat |
Inhibitors in the template | Re-dilute template after confirming ROX signal | ||
5 | Signal in Negative Controls (NTC) | Contamination in reaction components (e.g., DNA diluent) | Repeat with fresh Mix; prepare reactions in a laminar flow hood |
Cross-contamination or aerosol contamination | Implement strict lab zoning, use filtered tips, decontaminate workspaces, or replace consumables | ||
Residual fluorescent dye in instruments/PCR tubes | Clean equipment, perform background tests/calibration; use non-contaminated tubes | ||
6 | Abnormal Amplification Curves | High template concentration, degradation, or inadequate homogenization/dissolution; fluorescent dye degradation | Re-dilute after verifying ROX and multicomponent signals |
Evaporation (due to poor tube sealing) or pipetting-induced bubbles | Ensure tube integrity pre-run; inspect for bubbles; monitor post-run volume | ||
Instrument setting errors (e.g., incorrect baseline) | Adjust baseline (set endpoint to Ct−4) and reanalyze | ||
7 | Low Amplification Efficiency | Fluorescent dye degradation | Verify ROX signal; re-dilute |
PCR inhibitors in the reaction mix | Pre-dilute template to mitigate inhibitor effects | ||
Reference DNA dilution/pipetting errors | Ensure precise pipetting and homogenization (monitor ROX signal) | ||
8 | Excessive Amplification Efficiency | Non-specific amplification; excessive template concentration | Exclude highest-concentration wells; reanalyze standard curve |
9 | Irregular Amplification Curves | Inadequate reagent mixing | Homogenize thoroughly (check ROX signal) |
Inhibitors in the template | Re-dilute template | ||
Fluorescence signal interference | Confirm instrument compatibility with kit specifications | ||
10 | High CT Variability | Inconsistent threshold settings or instrument disparities | Standardize thresholds; compare data with validated controls (e.g., national reference standards) |
400-900-1882 
info@hillgene.com 
English
