Real-time PCR, also known as quantitative PCR or qPCR, has become an indispensible tool since its advent in 1992. The technique is constantly applied in various fields for measuring target gene expression, pathogen detection, DNA copy number measurements in genomic or viral DNAs, SNP (single nucleotide polymorphism) genotyping, verification of microarray results, drug therapy efficacy and DNA damage measurement. Real-time PCR is a refinement of traditional PCR; in real-time pcr, a relatively small amount of target DNA is amplified and detected through the course of the reaction with the help of fluorescent dyes. Obtaining accurate, consistent and reproducible results is a challenging task because of the varying factors that can cause discrepancies in measuring a given sample, and thereby affect the overall efficiency of the method. Because of its importance and widespread use, several companies offer real-time PCR platforms that meet the latest quantitative PCR applications.
Eppendorf’s Mastercycler® ep realplex is an instrument that exceeds the demand for rapid, accurate and reliable results. In our laboratory, we use it routinely for quantitative mRNA expression studies, verification of microarray results and pathogen (mycoplasma) detection in eukaryotic systems. It is a compact, easy to use and flexible platform. The compact design makes it an ideal instrument to work with in a big lab setting where space is a limiting factor. The time-efficient feature of the platform is another bonus factor for a packed lab where it is used often.
The results obtained through the Mastercycler ep realplex are consistent, reproducible and accurate. It’s advanced, high-speed gradient thermal block design makes the ramp heat up fairly fast and allows for a temperature gradient option. Because of its high speed, we perform as many as 5 runs a day in our lab, with reliable and accurate results. The realplex works well with all intercalating dyes or probe-based (SYBR® green, TaqMan®) detection formats making it a highly sensitive detection system for measuring target gene expression. The cycler works efficiently with both homemade qPCR mix, (i.e., mix made with reagents -- dNTPs, SYBR green, buffer -- from different companies) and commercially available kits. The platform is compatible with plates and reagents from different companies, which makes it an ideal and extremely flexible platform to use.
The one disadvantage is that the system needs to be calibrated for each new plate when it is used for the first time. It’s a bit noisy for the person sitting right next to it. For a novice, setting up the run in the software could be challenging, but once you get accustomed to it, it is pretty easy to handle. The software automatically saves each assay, which provides a back up for important data. The archiving of the data/assay by the software is another added feature of the platform. The technical service provided by the local representative was quick and helpful in situations that needed in-depth troubleshooting. In the three years of having used this platform, I have not come across any system failure problems. Overall, I give the platform five stars (on a scale of five) for its performance, design and reliability.