Reference Genes and Controls

Importance of PCR controls

A no-template control (NTC) allows detection of contamination of the PCR reagents. An NTC reaction contains all real-time PCR components except the template. Detection of a positive signal in an NTC reaction indicates the presence of contaminating nucleic acids.
A positive control may be necessary, for example, when amplifying a new target sequence to confirm whether the primer set or primer–probe set works. A positive control can be an absolute standard, which is a nucleic acid template of known copy number that provides quantitative information. Absolute standards, such as a nucleic acid from an established cell line, a plasmid containing cloned sequences, or in vitro transcribed RNA, are commercially available or can be generated in the lab. A positive control can also be a known positive sample, which is usually a substitute for an absolute standard and used only to test for the presence or absence of a target.

No RT control is a pivotal component of real-time RT-PCR setups, especially in gene expression analysis and viral load monitoring. This control, conducted without the addition of reverse transcriptase, is crucial for assessing the purity of RNA samples. Its primary function is to reveal the presence of contaminating DNA, which might otherwise be mistaken for RNA-derived amplification products. 

For viral load monitoring, a no RT control may be necessary, depending on the sample type and the life cycle of the virus species detected. Since reverse transcription cannot take place, a no RT control reaction allows detection of contaminating DNA, such as DNA from viral sequences integrated into the host genome. Contaminating DNA in RNA samples can be removed by DNase treatment before starting RT-PCR.

An internal positive control can be used to test for the presence of PCR inhibitors. A duplex reaction is carried out, where the target sequence is amplified with one primer-probe set, and a control sequence (i.e., the internal positive control) is amplified with a different primer-probe set. The internal positive control should be at a high enough copy number for accurate detection. If the internal positive control is detected, but the target sequence is not, then this indicates that the amplification reaction was successful and that the target sequence is absent (or at too low a copy number to be detected).

Several factors can generate a false negative result, such as errors in sample extraction or thermocycler malfunction. The most common cause is assay failure due to PCR or RT-PCR inhibition.

The most practical approach to control for the presence of inhibitors is to include an Internal Positive Control, or Internal Control (IC). This IC is simultaneously extracted and amplified (or only amplified) in the same tube with the pathogen target and should always be combined with an external positive control to prove the functionality of the reaction mix for amplification of the target. This combination rules out inhibition, among other malfunctions, and confirms that a negative result is truly negative.

Internal controls in PCR

Not all internal controls are the same (see table Features of internal controls), and each IC concept has value for specific applications. Endogenous ICs occur naturally in test specimens, such as a sequence of the host genome (e.g., ß-actin) or from normal microflora genomes (e.g., 16s). Exogenous ICs, on the other hand, are spiked into samples either during nucleic acid extraction or before PCR amplification.

Exogenous ICs can be homologous, where an artificial template is constructed with the same primer binding sites as the targeted pathogen sequence. Although the same primer set is used for target and IC, the sequence differs, enabling differentiation of pathogen and IC amplicons with different probes. Heterologous ICs, on the other hand, are designed with their own primers and probe.

Endogenous and exogenous homologous ICs carry the risk of impairing detection sensitivity for the pathogen target due to competition for reaction components. For example, a high starting amount of an endogenous IC template can impair assay sensitivity. This high starting amount can result from variations in the sample type or sampling technique. In the case of RNA applications, the high starting amount can also be due to enhanced expression levels of the IC due to disease-related cellular pathology. In the case of exogenous homologous ICs, using the same primers to amplify both target and IC leads to primer competition. Additionally, both endogenous and homologous ICs involve tedious IC design, and their use is restricted to a few applications or even individual assays.

In the context of process safety and workflow simplification, exogenous heterologous ICs are the most informative and flexible. The amount of IC template spiked into a sample is defined and consistent, and unrestricted design options enable optimization of IC properties. Only heterologous ICs allow for a design and setup that prevents competition for PCR components, and heterologous ICs are suited as universal controls, thereby making their implementation in new assays easy.

Features of internal controls
Feature Exogenous
Universal use in
multiple assays
No Yes No
Serves as control for
purification procedure
Yes Yes Yes
Differentiates purification
errors from amplification
Yes Yes No
Template quantities are
defined and consistent
Yes Yes No
Non-competitive internal
control design
No  Yes Yes