Misconception 1: The more instrument channels, the better

With the mature application of PCR technology, multiplex amplification has become increasingly popular, and fluorescent quantitative PCR machines are also not immune. From the single channel fluorescence quantitative PCR instrument launched by ABI company at the beginning, to now that various manufacturers have launched 4-channel, 5-channel, or even 6-channel fluorescence quantitative PCR instruments, the dazzling choices make people feel lost, and some people accidentally fall into the misconception of "the more channels, the better".

When using some manufacturers' 5-channel fluorescence quantitative analyzers, in order to ensure the accuracy and reliability of experimental results, ROX (a fluorescent dye) or Reference dye must be used in the experiment, and these fluorescent dyes must use a separate detection channel. In this way, there are only four effective channels that can truly detect multiple PCR fluorescence signals, and the use of calibration dyes may increase the cost of later use. Some fluorescent quantitative PCR detection channels are only open for their own manufacturers' fluorescent dyes or reagents, and there are not as many effective detection channels as claimed in promotional materials. It is particularly important to confirm the effective detection channel of the fluorescence quantitative PCR instrument before purchasing, and not just rely on advertising. When considering the number of channels for multiplex fluorescence quantitative PCR, it should also be based on the actual situation in the laboratory. multiplex PCR is not universally applicable or usable because it complicates the experiment.

Misconception 2: Real time PCR machines do not require gradient function

For quantitative PCR reactions using dye methods, although there are various PCR primer design software or empirical formulas to calculate the melting temperature (Tm value), the Tm value can vary greatly depending on the formula used and the primer sequence. The melting temperature of primers determines the annealing temperature. Moreover, the combinations of bases in the template are constantly changing. For special fragments, empirical formulas may not yield accurate results, and even slight differences in annealing temperature can have a decisive impact on the results. Therefore, "understanding the conditions" was once a headache. The emergence of gradient PCR partially solved some problems - the temperature control conditions of each well during the reaction process can be varied according to the gradient within a range, and based on the results, the reaction conditions can be explored in one step.

Not only annealing temperature, but also denaturation temperature and elongation temperature can be optimized - this is very important for most high fidelity Taq enzymes amplified by various polymerase mixed enzymes such as Invitrogen, Clontech, Promega, because there may be significant differences in reaction temperature between Taq and calibration enzymes, and optimizing elongation temperature is crucial.

The use of fluorescence quantitative PCR with gradient function can complete the optimization process that could only be achieved through multiple experiments in the past, simplifying the tedious experiments of exploring PCR reaction conditions, saving experimental time and improving efficiency, as well as reducing experimental costs.