Understanding the IPC in DNA Quantitation Assays

Carl Strayer
Promega Corporation
Publication Date: 2014
Many real-time quantitative PCR assays like the Plexor® HY System include an internal PCR control (IPC) to test for the presence of PCR inhibitors in the DNA samples. The basic principles of the IPC are simple enough, but effective use of this control in your DNA analysis workflow requires a deeper understanding of how the IPC works and how to interpret the IPC results. In the following discussion I will highlight important considerations to help you get the most out of your IPC results.

Principles and Limitations of the IPC

Typically, the IPC consists of a unique DNA template (i.e., one not found in a test sample) and a pair of specific primers, which are included in the reaction mix along with the specific primers for the primary assay targets. Amplification of the IPC should occur with equal efficiency (i.e., similar Cq values) in all samples and controls; if the IPC Cq of a test sample is substantially delayed relative to that of a standard or positive control, then this indicates the presence of an inhibitor in the test sample.

Ideally, the IPC serves two functions in the DNA workflow: 1) a means to assess reliability of the multiplexed DNA quantification assay(s) (i.e., flag a potential false negative or erroneously low quantification value) and 2) a way to help predict success of the downstream STR amplification reaction. Note that IPC results are more informative in the first application, and there are more pitfalls in the second application. There are several reasons for this.

First, it’s important to recognize that every PCR assay is unique. The reaction buffer composition, primer and probe design and configuration (e.g., sequence, concentration, amplicon size), and other assay-specific parameters uniquely influence a reaction’s susceptibility to PCR inhibitors. Within one multiplex assay, each component assay (target/primer/probe set) may have a different susceptibility to inhibitors. The IPC component of the quantification multiplex is generally designed and verified to be the most sensitive to inhibition: the proverbial “canary in the coal mine”. You don’t want a situation where the human DNA quantification components of the multiplex are inhibited but the IPC flag is not raised. However, while it’s reasonable to assume that the IPC results are relevant to the other components of a multiplex qPCR assay, it is another matter to assume that the IPC results of a quantification assay are equally relevant to the myriad STR assays available, as discussed below.

The second important consideration is the DNA sample volume in the quantification assay, which is usually fixed and at a low volume relative to the total reaction volume (the volume ratio). For example, in the Plexor® HY assay, the recommended sample volume is 10% of the total reaction volume (2μl of sample in a 20μl reaction), whereas many STR assays allow sample volumes of up to 75% of the total reaction volume. In this case, the concentration of any residual inhibitors in the DNA sample would be increased in the STR reaction by 7.5-fold, possibly beyond the tolerance threshold of the STR assay. Significant differences in the sample volume ratios between the quantification and STR assays makes the IPC less valuable as a predictor of STR amplification success.

Practical Tips for Using the IPC Results

We can apply these concepts to the implementation of the IPC assay in the DNA analysis workflow:

1. Know your assays.
Check developmental validation papers for the DNA quantification and STR assay(s) that you are using or intend to use. What are the reported inhibitor tolerances? Consider including inhibitor tolerance testing in both quantification and STR assay validations to determine the relative sensitivities to PCR inhibitors in your lab. If the STR assay is more sensitive to inhibitors than the DNA quantification assay, the IPC results will be less useful in predicting downstream amplification success.

2. Know the implications of a passing IPC result.
If the IPC passes, the quantification results are likely to be accurate. However, even if the IPC result is good (i.e., no flag is raised), success in downstream STR amplification is not guaranteed. If the DNA quantification value is low and a large sample volume must be used in the STR reaction, then it’s still possible for that reaction to be inhibited. For example, you determine the inhibitor tolerances of the two assays are equal—no inhibition at a final concentration of ≤500µM hematin. If a sample contains 1,000µM hematin and the quantification assay sample volume is one-tenth the final reaction volume, then you should see no effect since the final concentration of hematin would be 100µM. However, if you then add the same sample at 3/5 the final reaction volume of the STR assay, then the final concentration would be 600µM hematin, which would exceed the inhibition threshold. If that occurs, you may need to repurify the remainder of the DNA sample to remove inhibitors and possibly concentrate the DNA to reduce the volume required in the STR amplification.

Even if similar sample volume ratios are used in the quantification and STR assays, comparable success depends on comparable resistance to inhibitors. Knowing the relative sensitivity of your assays to inhibitors will help you make better decisions about the success of downstream STR assays.

3. Know the implications of an IPC flag.
If the IPC is flagged, then you can be fairly confident that there is real reason for concern, and action should be taken as a result. The quantification result should not be trusted, and the downstream STR assay may be negatively affected. What you do next depends on the situation:

If no concentration value was obtained for the sample, then this may be a false negative due to the presence of inhibitors. DNA should be repurified from the original starting material, then an aliquot should be re-amplified in the quantification assay.

If a concentration value was obtained for the sample and is sufficiently high to allow dilution, then it may be possible to dilute out the inhibitor. An aliquot of DNA can be diluted and re-amplified in the quantification assay. Using the IPC result and new quantification result from this second amplification, you can decide whether to proceed to the STR reaction or repurify the remaining DNA sample.

Finally, while recognizing that real-time PCR is usually used quantitatively, you should consider the IPC as a qualitative test. If the IPC flag is raised, the DNA sample should be considered unsuitable for further assays as is. The shift in the IPC Cq should not be used quantitatively to infer the amount of inhibitors present in the DNA sample nor to gauge the volume of DNA sample that the downstream STR amplification reaction may tolerate.

The IPC can be a useful tool but only if it is understood and used appropriately.

How to Cite This Article

Scientific Style and Format, 7th edition, 2006

Strayer, C. Understanding the IPC in DNA Quantitation Assays. [Internet] 2014. [cited: year, month, date]. Available from: https://www.promega.com/resources/profiles-in-dna/2014/understanding-the-ipc-in-dna-quantitation-assays/

American Medical Association, Manual of Style, 10th edition, 2007

Strayer, C. Understanding the IPC in DNA Quantitation Assays. Promega Corporation Web site. https://www.promega.com/resources/profiles-in-dna/2014/understanding-the-ipc-in-dna-quantitation-assays/ Updated 2014. Accessed Month Day, Year.

Contribution of an article to Profiles in DNA does not constitute an endorsement of Promega products.

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