This is part two of our “Let’s Talk about Real-time PCR” post, so check out part one if you haven’t yet.
Are you seeing strange or unexpected results in your quantitative real-time PCR (qPCR) reactions? Here are some commonly experienced issues, and some possible solutions to try.
Why is my PCR efficiency so low?
The efficiency of a PCR reaction is the fraction of target molecules copied every PCR cycle. In general, an efficiency of at least 90% is recommended. Many factors can contribute to low PCR efficiency, including
- Inefficient annealing between the primers and target,
- The primers binding to competing sites,
- Presence of inhibitors in the sample, and
- Insufficient reagents in the mix.
Double check the primer sequences to make sure there are no potential competing reactions. Run a melting curve to make sure you aren’t amplifying unexpected products. Reaction conditions may need to be optimized.
My PCR efficiency is too high! How is it possible to have an efficiency greater than 1?
If your PCR efficiency is high, or greater than 1, this would suggest that more than 100% of the targets are replicated each cycle. What’s going on? The presence of inhibitors in the sample is frequently the source of efficiency measures greater than 1. The greatest inhibition is in the most concentrated samples used in a dilution series, so the effect of inhibition on the standard curve is more pronounced at one end and distorts the slope of the curve, changing the efficiency calculation. To fix high PCR efficiency, you may need to dilute the sample to dilute out any contaminants.
Why are my Ct values so low? There's no way there was that much target in my sample.
Your Ct values are low because your samples may have evaporated if they were not stored correctly, which increases the concentration of the target. To fix this, carry out a melting curve at the end of PCR to make sure you are only amplifying the expected target and not amplifying something unexpected or primer dimers.
Why is there amplification in the no-template control?
A reason there is amplification in the no-template control could be due to contamination. Contamination is a major consideration when carrying out PCR. Make sure your pipettes and workplace are clean so you are not potentially transferring amplified products from a previous experiment into your solutions. Also, run a melting curve to see if you are amplifying primer dimers.