qPCR Troubleshooting guide

Sometimes no matter what precautions are taken, experiments don’t go as planned. Embrace the challenges of qPCR. On this page, we explore 5 common qPCR issues how they happen and provide insight into solutions we’ve learned through experience. 

Bookmark this page as a reference guide for anytime you run a qPCR experiment so you can always have these tips handy! qPCR Troubleshooting Guide (Click to download the PDF)

Click on the qPCR issue you're experiencing to see how to solve it

Problem: Incomplete blocking

A common cause of poor reaction efficiency is the presence of PCR inhibitors.

  • If poor efficiency AND an R2 value of < 0.98 are observed, likely culprits are pipetting error or the standard curve was not prepared fresh.​

How to avoid incomplete blocking

  • PCR inhibitors can be diluted away from a sample.

    Dilute the template prior to generation of the standard curve to find an ideal Ct range specific to your primer pair.

  • Become proficient at pipetting.

    Prepare each sample in technical triplicate, and if using a multichannel pipette, verify by eye that each sample is drawn up identically. Ensure a standard curve/dilution series is prepared fresh, because stored samples can evaporate over time.​

Problem: Unexpected values

Particularly when using a shared instrument, other users may have made changes to the protocol you’re using.

  • Other potential causes include incorrectly labeled samples or incorrect detection/selection of wells prior to the qPCR run.​

What to do when you see unexpected values

  • Check your thermal cycling conditions before the run to ensure the existing protocol is correct.

  • Verify correct dyes, volume, and wells are selected for detection by the instrument.

  • Setup a specific user account for each lab or group using the instrument to store saved protocols.​

  • Use a qPCR instrument that is capable of saving multiple protocols.

    The Azure Cielo System is a qPCR instrument with the option of 3 or 6 channels that stores customizable protocols.

Problem: Inconsistency amongst biological replicates

Inconsistency from sample to sample could be a sign of RNA degradation or minimal starting material.​

What to do when you see inconsistency amongst biological replicates

  • Prior to reverse transcription, check your RNA concentration and quality with a spectrophotometer and/or run your RNA out on an agarose gel.

    Anything less than the ideal 260/280 ratio of 1.9–2.0 could indicate the presence of PCR inhibitors, and a smear instead of two (2:1) bands on a gel indicates degradation.

  • Repeat the RNA isolation again.

    Consider using a method more suited to your needs, e.g. silica spin column vs phenol-chloroform.

Problem: Ct values are too early

Solving this issue requires some research into your existing primer pair. Check to see if any of these conditions apply to your run:

  • The primers may not span an exon-exon junction or could be generating more than one product.

  • The transcript might be naturally highly expressed in your samples.

  • If the samples have been stored for some time, evaporation may have caused an increase in the concentration.

Solutions

  • By ensuring your primers span an exon-exon junction, the chance for genomic DNA contamination is minimized.

    However as a preventative step, it is best to DNase treat the samples prior to reverse transcription.

  • To verify only one product is produced, BLAST the primers to determine hits within the organism.

    Include a melt curve at the end of thermal cycling, and/or run the qPCR products out on a gel.

  • If the transcript is naturally highly expressed, use the same dilution factor across all samples to dilute the template, yielding an ideal Ct range.

  • To prevent evaporation from samples, ensure tube caps are sealed with parafilm for long-term storage.​

Problem: Amplification in the no template control (NTC)

When pipetting template into wells that already contain a master mix, the template can sometimes splash into an adjacent NTC well.

  • Additional causes include reagent contamination or primer-dimer formation

How to avoid amplification into control wells

  • Clean your work area and pipettes with 70% ethanol, using 10% bleach if reagents have spilled.

  • Prepare a fresh primer dilution and be extremely cautious when pipetting the template (to prevent splashing).

  • Separate the NTC from any template samples on the plate as best as possible and use new reagents if necessary.

  • To detect primer-dimer formation, add a dissociation curve (melt curve) at the end of qPCR cycling and look for the presence of an additional peak, typically at a lower T.​​

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