A "Referral from the Doctor" Blog Article-
Quantitative real-time PCR (qPCR) is based upon the fractional cycle number at which a replicating sample of target DNA accumulates sufficient fluorescence to cross an arbitrary threshold. The threshold is either manually selected or auto-selected to fall several standard deviations above baseline fluorescence and below the plateau phase, where the amplification begins to attenuate. Typically, the threshold is adjusted to the mid-point of the exponential phase of the PCR, at a location suitable for all samples in the experiment. The CT (cycles to threshold) value for a given reaction is defined as the cycle number at which the fluorescence emission intersects the fixed threshold.
What if your assay is non-exponential?
The first step in qualifying newly-developed primers and probes is to test the assay on carefully quantified controls. Irregularities in the amplification of these controls indicate that some modification to the assay must precede its use upon valuable samples. The diagrams and statements below represent a few of the more commonly occurring issues and how they may be addressed by considering the information presented. This information is intended to be a starting point for trouble-shooting efforts and is not comprehensive. With regards to protocols, it is always best to follow the recommendations of the manufacturer of the polymerase enzyme. When multiplexing, use a master mix specifically designed for multiplex reactions.
Observation: Exponential amplification in the no template control (NTC)
Potential Causes: Contamination; carried over from reagent manufacture or possibly laboratory exposure to the same target sequence, as in gel electrophoresis
Corrective Steps: Clear work area with 10% Bleach and nuclease-free water; order new reagent stocks; relocate reaction set-up to a clean lab
Observation: Looping of data points during early cycles; high noise at the beginning of recorded data
Potential Causes: Baseline adjustment includes too many cycles; too much starting material
Corrective Steps: Reset Baseline to 3 cycles before the first indication of amplification
Observation: Unusually shaped amplification; irreproducible data; later than expected CT value
Potential Causes: Poor efficiency during PCR reaction; Difference in primer Tms is > 5 °C producing unequal extension; annealing temperature is too low; unanticipated variants within the target sequence
Corrective Steps: Re-design primers to a different region of the target sequence; keep melting temperatures within 2 °C of each other; keep the GC content to between 30%-50%; test assay performance against carefully quantified controls
Observation: Slope of standard curve is more or less than –3.34 and R2 value is less than 0.98
Potential Causes: Inaccurate dilutions; standard curve exceeds the linear range of detection
Corrective Steps: Recalculate the standard concentration or gene copy number using a spectrophotometer or other means; make new stock solutions of the control standards; eliminate extreme concentrations and limit range to 5 logarithms; consider using a carrier such as a yeast tRNA in the buffer used to generate the dilution series
Observation: Plateau is much lower than expected
Potential Causes: Limiting reagents; degraded reagents such as the dNTPs or master mix
Corrective Steps: Check calculations for master mix; repeat experiment using fresh stock solutions
Observation: Data values all unexpected
Potential Causes: Samples run out of order; plate inserted backwards; poor primer specificity
Corrective Steps: Re-run samples or plate using extra caution when loading; re-design primers to increase specificity
Observation: Actual CT is much earlier than anticipated
Potential Causes: Genomic DNA contamination; multiple products; high primer-dimer production; poor primer specificity; transcript naturally has high expression in samples of interest
Corrective Steps: DNAse-treat before reverse transcription; re-design primers to increase specificity; decrease primer concentration; increase annealing temperature; increase ramp rate; test assay performance against carefully quantified controls
Observation: Jagged signal throughout amplification plot
Potential Causes: Mechanical error; buffer-nucleotide instability; poor amplification or weak probe signal
Corrective Steps: Contact equipment technician; warm master mix to room temperature and mix thoroughly before use; allow primers and probes to equilibrate for several minutes at room temperature before use; mix primer/probe/master solution thoroughly during reaction set up; if the amount of probe is low or the signal too weak, the program will magnify the baseline noise; redesign the probe and primer sequences
Observation: Technical replicates are not overlapping and have a difference in CT values > 0.5 cycles
Potential Causes: Pipetting error; insufficient mixing of solutions; low expression of target transcript resulting in stochastic amplification
Corrective Steps: Calibrate pipettes; use positive-displacement pipettes and filtered tips; mix all solutions thoroughly during preparation and during use; hold pipette vertically when aspirating solutions-sterile technique does not ensure reproducibility when working with small volumes
Observation: Irreproducible comparisons between samples
Potential Causes: Efficiency of amplification is below 88% in one or both samples; differences in efficiency are > 5%; RNA degradation; inaccurate dilutions
Corrective Steps: Re-design primers for one or both genes; repeat experiment with fresh reagents and sample
Observation: No data in selected wells
Potential Causes: Wells not selected for analysis; wrong dye selection for analysis; failed first strand synthesis; no expression of target transcript
Corrective Steps: Check settings for data collection and for data viewing; repeat experiment with new reagents; test assay performance against carefully quantified controls
Observation: Lower concentrations all overlap
Potential Causes: Limited linear range of detection due to low expression of target transcript; carry-over contamination is obscuring the assay’s limit of sensitivity
Corrective Steps: Eliminate lowest concentration from the dilution series; create a standard curve that spans a higher concentration range
Observation: Highest concentrations overlap
Potential Causes: Limit of detection range
Corrective Steps: Eliminate highest concentrations from the dilution series; create a standard curve that spans a lower concentration range
Observation: Baseline drift
Potential Causes: Degradation of the probe
Corrective Steps: Set software for baseline subtraction; remove DTT from your reverse transcription step
Written by: Christina Ferrell, Ph.D., Technical Applications Specialist
