The efficiency of any PCR can be evaluated by performing a dilution series experiment using the target assay. Applied Biosystems recommends a 10-fold dilution series. After properly setting the baseline and threshold, the slope of the standard curve can be translated into an efficiency value:

Slope of standard curve indicates PCR efficiency. The StepOne™, StepOnePlus™, and 7500 Real-Time PCR System version 2.0 software programs calculate the efficiency of the PCR if a standard curve has been generated.
 
The efficiency of the PCR should be between 90–100% (−3.6 ≥ slope ≥ −3.3). If the efficiency is 100%, the CT values of the 10 fold dilution will be 3.3 cycles apart (there is a 2-fold change for each change in CT). If the slope is below –3.6, then the PCR has poor efficiency.

Parameters that affect the efficiency of PCR

Several parameters can affect the efficiency of the PCR (ranked from most to least frequent, based on our observations at Applied Biosystems Support):

  • Your samples may contain PCR inhibitors
  • Your PCR primer and/or probe design may not be optimal
  • Inaccurate sample and reagent pipetting
  • The standard curve may not have been properly analyzed

Your samples may contain PCR inhibitors

RNA that contains inhibitory compounds (e.g., sample preparation reagents, excessive protein) can lead to partial or complete inhibition of downstream PCR. PCR inhibitors originating from the starting material include heparin (>0.15mg/mL), proteins such as hemoglobin (>1mg/mL), polysaccharides, chlorophylls, melanin, humic acids, etc. Contaminants from the nucleic acid extraction phase include SDS (>0.01% w/v), phenol (>0.2% w/v), ethanol (>1%), proteinase K, guanidinium, and sodium acetate (>5mM).

How to identify PCR inhibition

Analyze RNA samples with a UV spectrophotometer, bioanalyzer or Nanodrop to assess quantity and quality. A high quality RNA sample should have an A260/A280 UV spectrophotometer reading close to 2. It has been observed that an A260/A280 reading of 1.8 suggests there is about 70-80% of protein in the samples; there are many proteins that inhibit both PCR and reverse transcription.

Inhibition plot: You can use real-time PCR data from standard curve plots to determine whether inhibition is occurring at a level that causes spurious results. When used to characterize inhibition, these semi-log standard curves are referred to as inhibition plots.  Please refer to the RNA Preparation and Reverse Transcription section of the Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR.

Solution

  • Perform RNA purification process on a sample using a new purification method. Choose your RNA extraction kit based on sample type. Refer to the RNA isolation kit decision tree to help make the right reagent/kit choice.
  • Further purify your samples: RNA with a significantly lower A260/A280 ratio should be further purified by phenol-chloroform extraction, LiCl precipitation, or washing to remove residual salt.
  • Test your sample at a lower template concentration at which it is known that PCR inhibition does not affect the real-time PCR results.

Your PCR primer and/or probe design may not be optimal

Optimal design of PCR primers and probe is mandatory for efficient PCR.

Custom TaqMan® Gene Expression Assay design is based on a template sequence provided by the customer. You can also design your own assays using other programs, and send the oligonucleotide sequences to Applied Biosystems for synthesis. You must perform bioinformatic evaluation of the sequences prior to submission to avoid PCR failure.

Read Bioinformatic Evaluation of a Sequence for Custom TaqMan® Gene Expression Assays to understand how to run a bioinformatics evaluation of a template sequence.

Bioinformatic evaluation allows you to accomplish three tasks:

  1. Identify a unique sequence by using BLAST (basic local alignment search tool). If the sequence submitted is unique (or from a non-homologous region), the assay that is designed on this template only detects the transcript of interest.
  2. Mask low complexity regions by using RepeatMasker. Low complexity regions, such as ALU sequences, are usually found within genomic DNA sequences. If you design your assay over these regions, the primers or probe will be depleted quickly if there is any genomic DNA in the samples.
  3. Mask SNP sites in the sequence, so that primers and probes will not be designed over SNP sites.

Inaccurate sample and reagent pipetting

Accurate pipetting with regularly calibrated pipettors is critical to obtaining accurate and precise data. Low volume pipetting (i.e., <5 µl) can contribute to imprecision, and pipetting of volumes less than this is not recommended unless the pipettors are designed for these low volumes and are regularly calibrated. It is also recommended to briefly spin down the sealed plates prior to running on the instrument, using low speed centrifugation. The table below lists some of the consequences of inaccurate pipetting.

Consequences of inaccurate pipetting

Pipetting problem Consequence
Sample: Poor pipetting of identical replicatesHigh CT standard deviations
Standards: Poor pipetting of standardsHigh CT standard deviations (identical replicates), R2 value <0.99
Standards: Consistent pipetting of excess diluent in serial dilution (e.g., 100 µL instead of 90 µL)Potentially good R2 value ≥0.99; however slope of standard curve will be inaccurate; perceived lower PCR efficiency of assay
Standards: Consistent pipetting of insufficient diluent in serial dilution (e.g., 80 µL instead of 90 µL)Potentially good R2 value ≥0.99; however slope of standard curve will be inaccurate; perceived higher PCR efficiency of assay
Standards: Consistent pipetting of excess standard sample in serial dilution (e.g., 12 µL instead of 10 µL)Potentially good R2 value ≥0.99; however slope of standard curve will be inaccurate; perceived higher PCR efficiency of assay
Standards: Consistent pipetting of insuficient standard sample in serial dilution (e.g., 8 µL instead of 10 µL)Potentially good R2 value ≥0.99; however slope of standard curve will be inaccurate; perceived lower PCR efficiency of assay

The standard curve may not have been properly analyzed

Solution

Once you have performed PCR, you must analyze the PCR products. We recommend that you use the Auto CT functionality to carry out the analysis.

Check if the NTC is negative or negligible.

The alignment of the experimental points on the standard curve must be verified. The replicates must be of good quality (CT values must be as close as possible, within 0.3 CT). Use the value of R2 to evaluate the quality of the results—a value of 0.99 and higher indicates good precision. The value of the slope should be as close to –3.32 as possible.

Ensure that the baseline is properly set. Use Auto CT or Auto Baseline features.

Verify the absence of outliers.

Low CT outliers:

If the point representing the most concentrated sample of the standard curve occurs at a later CT value than expected, but the rest of the curve is as expected, it is likely that this dilution point is showing inhibition (see figure below). Omit the point and reanalyze. This normally leads to what appears to be an over-efficient PCR (slope greater than –3.3, such as –2.9).

 

Dilutions (1:10) of a target, Exon 4, amplified in duplicate. The ΔCT between the two most concentrated samples is only 2.8, which indicates PCR inhibitors are present in the sample. Further dilution increases the ΔCT between consecutive dilutions to 3.3.

High CT outliers:

High CT outliers are generally the result of very few copies initially present in the reaction. They are ordinarily seen at CT values above 35 cycles and can increase or decrease the slope. These stochastic variations are inherent to the Poisson distribution, homogeneity of the mixture, and/or pipetting technique, and can constitute the detection limit. Some people consider the limit of quantification to be reached when the standard deviation of a standard is ≥ 0.5.

 

Variation in CT is greater for low copy targets. Consistent replicates for CT are obtained when there are 10 copies of target, but a spread is observed in the CT for replicates in which there is only one copy of target.