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Real Time PCR (qPCR) Troubleshooting

Introduction

Real-time PCR (qPCR) runs are complex experiments, involving a variety of different steps - from DNA or RNA extractions to cDNA synthesis, to precise sample preparations and complex quantification methods. With so many things involved, it's possible that something might go wrong down the line.

If this is the case, have no fear! This ZAGENO troubleshooting guide is here to help.

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Abnormal Curve Shape

  • The baseline of your instrument is too low: Calibrate the baseline according to the manufacturer guidelines
  • High fluorescence background: Be sure to mix your samples properly. If you are using SYBR® Green, try switching the intercalating agent.

Amplification on Negative Controls

  • DNA Contamination in your controls: The controls have been contaminated with the samples of another source.

Poor or No Amplification

  • Degraded template or oligonucleotides: DNA can be degraded after long-term storage, if stored at low concentration or at room temperature
  • Non-optimal amplification program: Your qPCR program may have a setting that does not allow for the amplification of your template. Make sure that the denaturation, annealing, and extension steps are sufficiently long for amplification
  • Poor quality probes: If you are using labeled DNA probes, make sure that these have not been through freeze-thaw cycles and have been stored at –20°C.

Poor Reproducibility Between Samples

  • Imprecise pipetting: The samples do not contain the same reagents due to imprecise pipetting. If you are using an automated pipetting system, ensure that the machine is properly calibrated
  • Primer design: Some primer sequences are sensitive to temperature. Be sure to check the annealing temperature of your primers

If you plan on repeating this experiment, make sure to include these controls to make troubleshooting easier

  • Mix the reagent well before using them
  • Use the same sample processing methods to compare your qPCR results
  • Perform a standard curve for every new primer pair to check efficiency
  • Make fresh dilutions for your reagents
  • Prepare your samples and run your qPCR in a different room, and change gloves frequently, to avoid contaminations
  • Use reference genes with similar amplification dynamics
  • Optimize your qPCR protocol every time you analyze a new target
  • Run a negative control experiment to measure the background of your DNA dye
  • Always use the same method of quantification to compare your gene expression levels