James Netterwald, PhD, is a freelance science and medical writer based in New Jersey. His writing serves every life science industry.
Real-time qPCR is one of the most important tools to emerge from biological sciences in the last 20 years. By enabling the accurate quantification of gene products in real time, this technology has revolutionized the study of genomes, leading to the continuing evolution of genomic medicine, bioinformatics, clinical diagnostics and more. In addition, an ever-expanding array of applications have been derived from or improved by the development of real-time qPCR. These applications include melting curve analysis for identification of mutations and determination of GC content, quantification of gene expression, quantitative chromatin immunoprecipitation (ChIP), single nucleotide polymorphism genotyping, determination of copy number variation and more.
What should I consider first?
Before choosing a thermal cycler, you should first consider your experimental approach. For example, does your approach require you to select a dye or probe chemistry? Will you need to perform singleplex or multiplex reactions? How many reactions will be run in a single experiment? How many different colors do you want to measure in a single reaction—one or two or six? These are a just a few design considerations that directly impact the decision to purchase any given real-time thermal cycler. Ultimately, you should choose a system that is in line with your experimental design.
How important are throughput and speed?
Another consideration to make when selecting an instrument is the throughput of the thermal cycler. The number of wells on the instrument dictates throughput. Real-time thermal cyclers can load a wide range of reaction tubes in their heating blocks, ranging from 96 wells to more than 1,000 wells.
Another consideration is the speed or cycling time of the instrument. Some instruments can complete a single run in 30 minutes, but others may take 60 minutes or more. The cycling time depends on the user settings for temperature and time per cycle. However, when comparing two different thermal cyclers that are running the exact same program, the instrument that changes block temperature fastest will generally have the shortest run completion time.
What about the sensitivity of detection and dynamic range?
So now that you have considered your experimental design as well as the throughput and speed of the system, it’s time to consider the sensitivity of the instrument. Some instruments can detect as low as one copy of a gene or gene product. These are the most sensitive instruments. The sensitivity of the instrument also dictates its signal-to-noise ratio, which is the ratio of fluorescence detected from a specific hybridization event with the nucleic acid of interest to the amount of fluorescence from nonspecific hybridization events. In other words, the higher the signal-to-noise ratio of a real-time thermal cycler, the higher its sensitivity.
Related to the sensitivity of a real-time qPCR method is its dynamic range, which is typically defined as the range of initial template concentrations over which accurate threshold cycle (Ct) values are obtained. The dynamic range of detection is usually on the order of several logs, and the larger the dynamic range, the greater the ability to detect samples with high and low copy number in the same run. You should avoid extrapolating initial template copy number values beyond the dynamic range.
Do I need to use a reference dye?
Another consideration to make before buying a thermal cycler is whether or not it requires the use of a reference dye. The design of some thermal cyclers causes well-to-well variation in detection. One common way to compensate for this variation is by adding a reference dye, such as ROX dye, to each reaction vessel because the concentration of the reference dye does not vary during PCR; therefore, the fluorescence of the dye is constant throughout the run and from well to well.
If the system you are considering does not require a reference dye, check to see what detection method it uses. If well-to-well variation is an issue, determine how the system compensates for it. Although using a reference dye does not necessarily produce better quality results, workflows that use dye differ slightly from those that do not include dye. The bottom line is to be aware of the workflow required by your system so you are sure it will work for you.
Should I automate my workflow?
Finally, you need to decide if you want the run to be automated or manual. In other words, do you wish to add the samples and consumables to each reaction tube in preparation for a run, or do you want a liquid handler to do it for you? Thermal cyclers can be coupled with an automated liquid handling system that uses a robotic arm to perform the reaction setup. As expected, these instruments come with the added bonus of being able to analyze more than 10,000 reactions in a single run.
Summary
Purchasing a real-time thermal cycler can be a simple decision if you have a clear application in mind and a clearer understanding of the detection chemistry required for the application. After the detection system has been selected, determining the sensitivity, cycle time and throughput required in your thermal cycler should be a breeze.
The image at the top of the page is from Roche Applied Science's LightCycler® 96 System Performance Data.