Observe Quantitative Replication of DNA in Real Time with qPCR System

Quantitative real-time PCR, or qPCR, has quickly become a staple in research institutions. While conventional PCR allows for the qualitative measurements of replicated DNA, qPCR also offers the researcher the ability to observe, in real time, quantitative replication of the DNA with the use of fluorescent dyes, giving much more accurate results than typical PCR. As the technology has become more widely adopted, qPCR equipment has undergone several advances.

PCRmax Eco 48 Real-Time qPCR System

PCRmax Eco 48 Real-Time qPCR Thermal Cycler

The PCRmax Eco 48 Real-Time qPCR System utilizes a 48-well plate that has the same geometry as one-eighth of a standard 384-well plate. A minimum volume of 5 μL is validated, resulting in more efficient use of expensive and difficult-to-acquire DNA. The smaller plate provides ±0.1 ºC temperature uniformity across the block. This also allows users to run duplicates as opposed to triplicates of their reaction, as required by a typical 96-well system due to poor temperature uniformity.

Fast, uniform temperature control is important because accurate dwell temperatures ensure that primers bind most efficiently and polymerase enzymes work optimally, generating the maximum yield of target DNA. The hermetically sealed hollow block contains a conductive fluid and two opposing agitators driven by electromagnetic motors. During PCR cycling, these agitators rapidly circulate fluid throughout the hollow block, transferring heat from the Peltier quickly and evenly.

While it may seem counterintuitive, the 48-well plate design allows users to run more reactions than a 96-well device. Thermal control in the Eco48 is more efficient—in traditional 96-well Peltier systems, thermal efficiency decreases as distance from the Peltier increases. With Eco 48, faster and more precise cycling boosts sample throughput because the need for multiple replicates is reduced by increased uniformity across the block. Well-to-well, plate-to-plate, and instrument-to-instrument results are as consistent as running a single experiment. For example, the software allows different experiments to be combined, that is, if the same experiment is being run on different Eco 48 systems, the results can be combined into one consistent experiment. High uniformity reduces the need for experimental replicates, which conserves samples, eliminates the need for more expensive instruments, reduces reagent running costs, and ensures that the entire plate of data is valuable.

48-well plate 96-well device run reactions

Fast sequencing is also achieved due to the technology used to read the plate. Adaptive LED control, referred to as ALC, can normalize fluorescence across all wells for every cycle run and provide specific tuning for each LED following a PCR cycle. LED adjustment following each amplification allows the instrument to reduce exposure time as signal intensity increases, maximizing the linear range of emission detection while minimizing lighting artifacts. Since only one image is required to cover the full range of the sample, processing time is reduced. Sensitivity is also increased as the exposure level is done per well by dye. There is no universal exposure setting that can lead to inaccurate results.

The Eco 48 is supplied factory-calibrated for use with SYBR, FAMTM, HEXTM, VICTM, ROXTM, and Cy5, and can also be used with any qPCR fluorophores overlapping with these calibrated dyes. Two software programs are included. Eco 48 Connect software provides instrument control, data collection, and data analysis. The most common qPCR parameters are automatically in place. These can be easily changed to meet specific experimental requirements. The drag-and-drop interface makes creating and managing experiments simple and easy.

Eco study software allows researchers to easily view the amplification plot, melt curve analysis, and analyzed results, including Cq values, PCR efficiency, R2, and y-intercept. Data can be exported into Excel or CSV and customer reports can be generated directly into PowerPoint or PDF formats. High-resolution images can be directly exported in multiple image formats, ready to use for publication submission or in a PowerPoint presentation. The device also conforms to MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, making data analysis and submission for publication review more efficient.

The software, combined with the accuracy of the instrument due to its precise temperature control, provides PCR efficiencies between 90% and 110% and R2> 0.99 when using standard optimized assays. This makes the Eco 48 suitable for the following applications:

Absolute and relative quantitation

  • Absolute quantification standard curves with efficiency calculation
  • Relative quantification using the ΔΔCq method with support for multiple reference gene normalization
  • Relative gene expression with efficiency control
  • Next-generation sequencing (NGS) library quantification
  • Copy number variation: insertions, deletions, inversions
  • RNA characterization: siRNA and miRNA
  • Viral load

Genotyping and high-resolution melting

  • Allelic discrimination
  • DNA methylation
  • SNP verification
  • Microbiology: pathogen detection and identification; high-resolution melting (HRM)
  • Genotyping, methylation studies and mutation screening.

PCRmax Eco 48 Real-Time qPCR graphs

While there will continue to be a place for a conventional thermal cycler on a laboratory bench for routine genotyping and quality work, real-time PCR is becoming almost as common. The ability to quantify the polymerase chain reaction in real time eliminates tedious and inaccurate quantification methods that are required after typical PCR and provides results that are highly repeatable and accurate. The method allows for immediate data analysis and advanced experimental design. As the technique has become more popular, reagent kits have also become more readily available, with many primers already created for a wide range of targets. This allows even scientists new to the field to run experiments with minimal training.

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