Since its first introduction in 1993, quantitative PCR (qPCR) assays have become a popular tool in molecular biology. Today, expression analysis and copy number qPCR assessment are heavily dependent on these modern qPCR systems. Besides advanced alternatives such as the ddPCR method, qPCR CRO has started offering solutions such as ddPCR gene expression services. However, similar to PK labs, qPCR CROs should focus on enhancing data quality and instilling confidence through reliable and reproducible results.
Despite the advantages of qPCR assays, the final experimental data obtained can be compromised due to a lack of quality control. Different control samples are a prerequisite in qPCR assays. The current article dives deep into the issues of quality controls and discusses effective quality control protocols and strategies for enhancing data quality in qPCR CRO services.
Effective quality control in qPCR assays
No template controls are critical to identify contaminant DNA. An amplification in the no template control sample is an indicator of potential contamination or the generation of primer dimers. However, researchers can ignore these signals if the difference between the highest Cq value sample and the Cq value among the no template control is sufficiently large. Notably, smaller differences between the unknown samples and no template controls should be avoided.
In qPCR gene expression analysis, the minus reverse transcription control is crucial for ensuring the absence of contaminated genomic DNA. Employing enzymatic DNA treatment to remove contaminated DNA is a promising strategy. However, this treatment is not 100% effective and may require a minus reverse transcription control. This control verifies the absence of DNA by performing a qPCR assessment of the target DNA on the crude DNA. Amplification in the minus reverse transcription control indicates primer dimer formation or potential residual genomic DNA contamination.
Having reference samples with non-copy numbers is a specific control measure for qPCR copy number analysis. This control sample can be a reference point while calculating and interpreting copy number results. Including several control samples will generate more reliable and accurate data. Besides, one may also include reference samples for qPCR expression analysis. These reference samples can be biological control agents.
Must Read: Optimizing Accuracy in qPCR Expression Analysis: Quality Assurance Strategies
Researchers can include two additional controls, namely internal amplification controls and no amplification controls when working with probes. The no amplification controls contain all components and the template except the polymerase. An amplification may suggest autofluorescence, which may be an indicator of probe degradation. An internal amplification control helps remove false negative data due to inhibition or other issues with the qPCR reaction. Internal amplification controls are crucial for samples prone to inhibition as they can generate false negative results. Internal amplification control is a sequence of DNA that is spiked and co-amplified with the sequence of interest. If there is no amplification signal for internal amplification control, it may suggest a false negative result and can be correlated to inhibition or error in the qPCR reaction.
Furthermore, dilution control can be a great alternative to internal amplification control for detecting inhibition when using intercalating dyes. A diluted sample may reduce the effect of inhibiting substances. Hence, researchers can include diluted samples as a control to evaluate inhibition.
