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February 01, 2016 (Vol. 36, No. 3)

Improving Real-Time PCR Data Quality

Developing an Assay to Deliver Reliable and Reproducible Results with Novel Instrumentation

  • The polymerase chain reaction (PCR) has radically transformed biological science, allowing sophisticated analysis of genes and the genome. Revolutionizing the study of DNA, PCR is often hailed as one of the most important scientific advances of the 20th century. Over time, PCR has evolved into fluorescence-based quantitative real-time PCR (qPCR), which is now considered the molecular diagnostic technique of choice due to its capacity to detect and measure minute amounts of nucleic acids in a variety of samples from multiple sources.

    Due to its practical simplicity, in combination with its outstanding capabilities, including speed, sensitivity, and specificity, qPCR plays a huge role in a number of applications, among them gene expression analysis, microRNA analysis, single nucleotide polymorphism genotyping, copy number variation analysis, and protein analysis.

    The success and reputation of qPCR is reflected in the abundance of publications reporting qPCR data. Despite substantial advances in the accessibility and ease-of-use of qPCR for diagnostics, generating an assay that is capable of delivering reliable, reproducible, and meaningful results is still a challenging task.

  • Assay Development

    Click Image To Enlarge +
    Figure 1. Baseline corrected amplification plot showing the data from all 48 wells of the Eco 48 plate.

    The design of assays has progressed considerably, and today scientists can reference sequences from numerous publications or select from predesigned assays. However, there are still occasions when scientists want to target a different region entirely; therefore it is necessary to develop a de novo assay.

    Assay development is fundamental to qPCR, so it is important that it complies with Minimum Information for Publication of Quantitative Real-time PCR (MIQE) guidelines, and that overall quality has been rigorously assessed. When assays are poorly designed, performance suffers and scientists can experience poor signal generation, unexpected cycles-to-threshold (Cq) values, or data that cannot be reproduced. Recent reports have suggested that up to 90% of papers published in the biomedical research arena are not reproducible, relating to the reliability of the assay.

    The associated challenges of developing successful assays can be resolved by choosing PCR instrumentation that is both reliable and efficient. Temperature control is at the heart of qPCR, and without accurate and exact temperature control data accuracy can be distorted, and more replicates must be performed to improve confidence. The Eco 48 package from PCRmax is an ideal system for assay development and optimization. This is due firstly to the unique thermal block design of the Eco 48, which demonstrates the highest uniformity of any block, achieving ±0.1°C uniformity at 95°C. Secondly, the unmatched uniformity is recorded with no settle time, meaning that with the Eco 48, hold times can be shortened, ensuring all samples are immediately at the correct temperature and are completely uniform. The Eco 48 can easily achieve 40 cycle protocols in 40 minutes or less when optimized, using standard chemistries.

    To define and prove the thermal uniformity across the thermal block of the Eco 48, replicate samples were put through a thermally demanding protocol, High Resolution Melt (HRM). Each of the 48 wells had 1 × 108 copies of template in a 10 µL final volume. The plate was sealed and centrifuged for one minute at 1,200 rpm. The 40-cycle PCR step and HRM was performed in a total time of just 43 minutes with a temperature resolution of 0.1°C, after which the results were analyzed using Eco Study software to determine the Cq and the thermal melt (Tm) values for each of the replicates and the degree of uniformity across all replicates, as an indicator of block accuracy and, as such, data reproducibility.

  • Click Image To Enlarge +
    Figure 2. Normalized melt plot showing data from 48 wells of the plate.

    The amplification plot for all 48 wells was determined. Figure 1 demonstrates the precision of amplification across the entire plate. The 100 bp template was based upon Lambda phage DNA, and was amplified for 40 cycles (95°C, 10s; 60°C, 30s) using the GoTaq® qPCR Master Mix from Promega. The fluorescence data was collected at the end of the 60°C step using the green channel. Analyzing the data showed an average Cq of 13.31 (SD ± 0.061), equating to a %CV across the plate of just 0.46%.

    Measuring the Tm of a PCR run is one of the best measures of determining block uniformity. Following the amplification steps, the Tm was determined by running a High Resolution Melt. The 100 bp PCR product template was melted over the range 75°C to 95°C, and the Eco 48 measured and collected the fluorescence data with every 0.1°C of temperature change, which confers the accuracy required to detect class IV SNPs with greater than 99% accuracy. Figure 2 shows the normalized melt curve.

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    Figure 3. Summary of Cq and Tm data from each of the 48 wells of the plate.

    Additionally, the Tm average across all 48 wells was recorded as 84.45°C (SD ± 0.0058), equating to a %CV across the plate of just 0.07%. Figure 3 shows a summary of the results obtained.

    Fast, uniform temperature control is crucial to assay development and the qPCR process to ensure accurate quantification of any sample in any application. The Eco 48 from PCRmax removes multiple variables, ensuring the variation that is measured is due to the samples themselves, and not due to poor quality thermal control from the qPCR system biasing the data. The unique design specification of the Eco 48 block ensures that it is the most thermally accurate and uniform block based system currently on the market, which increases accuracy, reduces the need for numerous replicates, and improves the confidence in the data produced and the conclusions drawn from these data. 

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