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In this study, the SYBR Green-based Real Time PCR assay was applied to mcr-1, mcr-2, and mcr-3 genes. This assay can be performed in any laboratory with little to no experience. However, in order to detect the target amount accurately, the use of a Real Time PCR machine is necessary. Regardless of the method you choose, it is important to understand how the method works. Find out – zbadajkleszcza.pl/badanie-kleszcza-cena/ Minimized By Using A High-quality Template To calculate the level of gene expression, Real Time PCR uses a standard curve analysis technique. The results of RT-PCR can be compared against a known template. The template used in this method is either gDNA, cDNA, synthetic oligos, or plasmid DNA. The Ct value of the unknown sample is then subtracted from that of the known template (Ct value). This method is highly accurate, but it does not provide the highest level of information. One of the most significant factors influencing the reliability of real-time PCR is the template quality. Numerous authors have shown that the presence of inhibitory components significantly decreases the yield of real-time PCR assays. Inhibitors are either reagents used during the nucleic acid extraction or copurified components of biological samples. The high degree of inhibitory activity creates false-negative results. However, these problems can be minimized by using a high-quality template. The five-parameter Richards function is an alternative method for calculating the rate of amplification of a specific DNA fragment. Using the five-parameter Richards function, it is possible to fit the experimental fluorescence data to Eq. 3. Then, using the results of the PCR, one can find the tangent crossing point and the crosspoint between x and y axes. The results of this analysis are compared with those of the Cy 0 method.