Key parameters and important characteristics of fluorescent quantitative PCR instrument

When purchasing a fluorescent quantitative PCR instrument, several key parameters should be paid attention to:

1. The detection throughput of the instrument (Throughput)

The first thing to consider is the frequency with which the laboratory currently needs to use a quantitative PCR instrument. David Ginzinger, head of genome analysis equipment at the University of California, San Francisco Cancer Center, has tested most of the quantitative PCR machines on the market. He said that if it is not used in large-scale batches, for example, it is mainly used to test known genes, then expensive high-end products are really not needed. But if it is those laboratories that need to find new drug targets and disease markers, they need instruments that can accommodate 384-well plates and run faster. However, just such a high-throughput instrument is not fast enough, and you will find that sample preparation becomes a bottleneck for the speed limit. Therefore, high-throughput laboratories often need further automated instruments. As long as the "click", the instrument will automatically process samples, purify nucleic acids, and prepare PCR templates.

2. The ability of multiple amplification

Multiple amplification is getting hotter and hotter. Real-time quantitative PCR machines are not immune. The high-end quantitative PCR instrument has multiple detection channels, so the instrument can detect the amplification process of multiple templates in the same sample tank. The new LightCycler2.0 has 6 detection channels, 3 more than the original. SmartCycler II has 4 detection channels. Stratagene, famous for its library

products, also has a quantitative PCR machine Mx3000P, which is less than half the price of Roche and has a good reputation, which is 4 detection channels. However, multiple amplification is not suitable for everyone, because it complicates the experiment.

3. software

When you are going to choose a quantitative PCR machine, you must check the accompanying software. Is it easy to use? Is it possible to perform the analysis you need? There are several output formats. However, you need to pay attention to whether the software that is too easy to use has simple functions and cannot perform complex calculations. The previous software still requires you to do further analysis. The new software can already handle everything. In addition to basic functions, some software can also have more functions, such as primer and probe design, multiple analysis design, background correction, data normalization, and so on. Willian Demyan, head of the Application and Technical Services Department of Roche's Applied Science Department, emphasized: Quantitative PCR is a geometric amplification process. At the beginning, the difference between one copy and the result is geometrically different.

4. Flexibility

Although the large-scale busy laboratory equipment is advanced, it is enviable, and the conventional laboratory can also have wonderful choices. The current suppliers have many technical experts, not only understand your current needs, but also consider possible changes in your future requirements very thoughtfully, such as providing a variety of upgradeable accessories and modules. BioRad's MyCycler can select modules and upgrade to real-time quantitative. ABI's quantitative PCR instrument has a variety of different sample tanks, which can change a 96-well tank into a double 384-well tank to meet part of the high-throughput requirements. For laboratories that are just starting to do a small amount of qPCR, the SmartCycler II quantitative PCR instrument is a good choice. A basic module has 16 independent temperature-controlled sample tanks, and 16 completely independent different experiments can be performed at the same time or before and after. Access"; when the laboratory needs a larger-scale experiment, it can be upgraded economically, from 1 to 6 basic modules, that is, 96 sample wells. Although a 96-well plate cannot be used, when a student starts a small experiment, others can plug in and start their own independent experiments at any time without interfering with each other, which increases flexibility. It is a very flexible choice for laboratories that only do a few experiments at the beginning and have a tight budget.

5. Uncertainty

  Although the software can be partially corrected, it is better to buy an instrument with less system error and not require so many calculation corrections than to rely on calculation corrections. Cinzinger believes that special attention should be paid to the differences between the sample holes of the instrument. In addition to the well-known temperature difference between the sample holes, there are also the optical path distance between the sample hole and the detection probe, the slight difference in the length of the optical fiber responsible for receiving and transmitting signals, the slight difference in the distance and angle between the lens and the sample hole, and even The influence of the sample slot on signal transmission. Cinzinger has done the same test on most of the commercially available quantitative PCR machines. He performs real-time amplification of the same samples and reaction reagents in each well of the instrument. Unfortunately, many times, there will be different results between different sample wells.