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Sunday, March 24, 2013

Real-time PCR inhibition and poor reaction efficiency


Real time PCR efficiency is one of the critical factor in real time PCR assay development and optimization. A 100% efficient PCR result indicates a well optimized assay development. Below which indicates a need to further optimize the reaction parameters, which includes, to mention few, primer probe concentration, buffer optimization, etc. An accepted PCR assay efficiency ranges from 90% - 110%. A standard curve can be generated andefficiency can be calculated.


Real - time PCR: Causes of high or low efficiency



As mentioned earlier an accepted PCR assay efficiency range for real time PCR assay is 90% - 110%. An assay efficiency above 110% indicates a possible inhibition in the real time PCR reaction. The main reasons for inhibition in the reaction is the poor quality of DNA or RNA used as template, use of high template concentration. Sub-optimized extraction procedures to get the DNA or RNA purified, presence of high amount of chaotropic salts which can inhibit Taq polymerase activity. Low efficiency real time PCR assay is mainly due to the poor reaction conditions or reagent concentrations which includes sub-optimized concentration of Primers, Probes, Taq polymerase, Magnesium, etc. and the reaction condition includes improper or sub-optimal thermal cycling.

The problem with skewed efficiency in Real time PCR
Efficiencies outside the range of 90–110% may artificially skew results and lead to false conclusions, mainly because targets for comparison will have different efficiencies. In addition, inhibition and poor efficiency can affect assay sensitivity, leading to a smaller dynamic range and decreased versatility.

How to check the efficiency of real time PCR assay is skewed or not???
The best method to determine assay efficiency is to generate a standard curve of template diluted over the range of what will be encountered with the unknown samples and look at the efficiency over that range. It should be as close to 100% as possible. A dissociation curve or gel showing multiple peaks or products means there is a competition for reaction resources that almost certainly will have an effect on the reaction efficiency.

Resolution for Poor Efficiency or Inhibition of a real time PCR assay
Once it is found that the assay is not performing well as 100% efficiency or inhibition is occurring one should take steps to make the assay efficiency to an accepted range, one should strive to achieve an assay efficiency of 100%. The following steps can be used to get the desired assay efficiency.
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  • For inhibition, try to remove the highest concentration and analyse the standard curve, this may bring the assay efficiency close to the desired range. This is because of the fact that highest template concentration will also have the highest amount of inhibitors, while diluting inhibitors are also getting diluted to a level which is not inhibitory to the reaction.
  •  Re-purifying the template is another option this can also be done without much difficulty, use enough washing steps to wash off chaotropic salts, which can inhibit the reaction.
  •  Assay optimization is another solution for poor efficiency. It is a laborious process, it can increase the complexity of the assay.
  1. Varying Magnesium concentration can be accessed from a range of 3mM to 6mM which can improve the assay efficiency.
  2. Primer Probe concentration optimization also plays a major role. Primer dimer issues need to be resolved for better efficiency reaction and specificity and sensitivity. Sometimes primer need to be re-designed for better results.
  3. Thermal cycling optimization also plays major role, this needs to be adjusted based on the Tm values of the primers.
Following these should bring the real time pcr assay efficiency to be in the range of 90% - 110%.
Note:  a 100% assay efficiency yield a standard curve slope of -3.32.