Mastercycler® RealPlex² From Eppendorf

The Eppendorf Mastercycler® RealPlex² is a small, robustly designed instrument for real-time PCR. The machine features, like some other Mastercyler devices from Eppendorf, a special 96-well heating block, which enables rapid thermal cycling. Hence, the time it takes to complete an assay is decreased. In a normal working day in our laboratory, the RealPlex² can be run four to five times, depending upon assay type. A standard 40 cycle Taqman® assay can be run in approximately 80 minutes, including data analysis and report generation. The RealPlex² contains a maximum of two filters, which enables fluorescence monitoring at 520 and 550nm. Hence the machine is compatible with the most widely used reporting dyes (SYBR Green I, FAM, JOE & VIC). I also use the 520 nm channel to detect fluorescence from PicoGreen assays, for quantification of cDNA after reverse transcription reactions (which means I didn’t have to buy a separate 96-well plate reader).

Being a newcomer to real-time PCR, I trialled a few different instruments from various suppliers before buying. I found the RealPlex² to be the best of the bunch based on the software interface, cycling speed and general ease of use. With the Eppendorf software, it is very easy to program in an assay. Unlike other systems, the software doesn’t force the user to lay out the assay in a particular way. It is very flexible and lets you set things up exactly as you like. Programming dilution series is very simple and straightforward, which I found problematic using other instruments. During cycling, the data is displayed in an easy to read format, and the cycling can be paused at any time. At the end of the program, all experimental conditions are collated into a professional-looking report with just one click. Data are saved automatically after every run, in a flexible filing structure. The software allows each user to maintain their own data folder, making it quick and easy to find past experiments. The data archive can also be password protected for security.

Unlike many other qPCR machines, this machine uses 96 static LED’s to generate light for excitation. This means that the whole fluorescence detection process is static and involves no moving parts. While this may sound unimportant, I found that other machines which use a moving excitation & detection head are irritatingly noisy, especially when measuring PCR product melting curves with SYBR Green. The RealPlex² is very quiet and has a small footprint, so it’s ideal in a small PCR lab.

This RealPlex² is a good research tool for relatively simple real-time assays. Since it can monitor only two channels, it cannot be used for anything more complex than a duplex, two-color reaction. However, to date, my research has not given me reason to use more than two reporter dyes simultaneously. One possible drawback is that the machine cannot detect ROX (620 nm) reference dye, which is sometimes used to normalize starting fluorescence values. Applied Biosystem’s Taqman® Mastermix users should be aware of this. Researchers requiring more complex real-time assays, or a wider choice of fluorophores should consider the big-brother product, the RealPlex⁴. Personally, I would like the ability to take numerical fluorescence data off the machine, i.e. export it into a spreadsheet. I think this is lacking in the current software, but I am told that this may be remedied in coming software updates.

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Dr. Daniel Palmer
Collaborative Research Manager
Q Chip Ltd
United Kingdom