Tuesday September 20, 2005
Real-Time PCR is rapidly growing and as the number of equipment manufacturers has increased, the price of machines has subsequently decreased. Once seemingly unobtainable, real-time PCR is now available to most labs. The Quantica is Techne’s offering into the real-time market, consisting of a 96 x 0.2ml well, peltier block machine. The system uses a photomultiplier tube (PMT) and is capable of 470nm to 650nm excitation and 500nm to 710nm detection. It holds up to 4 filter sets at a time, with all the normal dye sets available, so it is possible to multiplex up to 4 dyes at once. The filter cartridges fit into the carrousel, enabling you to fit a new filter set yourself.
The first thing that struck me was the simplicity of use. The software for programming experiments is very straight forward. The experiment editor, which holds all information on each run, allows you to create a new program, load or edit an existing one. I found creating a new program very intuitive because as you enter the parameters, a graphical representation of the program is shown. Adding readings and disassociation curves are just as simple and, while the manual is excellent, it really isn’t needed since the software has been well designed. A useful button allows you to add 10 cycles during a run, you can look at the raw data coming off and add cycles if the reactions are not very active. This feature is handy when trying out new reactions.
Designing a plate layout is even easier; each type of sample is assigned a color (standard curve, house keeping genes, unknown, etc.), the number of replicates is set and the plate is filled. Entering and saving values for your standard curve is possible for use in later experiments either before or after running the experiment. Another useful feature is the ability to flip the plate 180 degrees, just in case you put the plate in the wrong orientation.
The software analysis wizard provides two options: 1) selection of analysis type (at each stage the wizard describes what each option does) or 2) skip to the end input the settings yourself. There are a number of options for calculating crossing points via the software or it can be performed manually. In addition, by clicking on single samples you can choose to view individual graphs or all reactions combined on a single graph. When using an intercalating dye such as SYBR green, which our lab employs, the software allows you to use the dissociation curve for genotyping. Again, you have the option of manually selecting the peaks or following a wizard. We have found the sensitivity of the machine to be very good, comparable with other machines we looked at. Each well is read individually so the read time is a little longer. However, this aspect also prevents cross-over between wells.
The Quantica system is a great value for the money, allowing the flexibility that most labs will need. It is simple to use, has a good user interface and provides an excellent analysis package, all at a very reasonable price. There is excellent training and technical support covering everything from the basics to primer design.
Matt Hall
Technician
Marine Biological Association of the UK