Usually, a primer concentration of 0.2μM can yield good results, and a reference range of 0.1-1.0μM can be used for setting. The concentration of the probe used is related to the fluorescent quantitative PCR instrument, probe type, and fluorescent labeling substance used. Please refer to the instrument manual or the specific usage requirements of each fluorescent probe for concentration adjustment during actual use.
Usually, the amount of RNA template is based on 10pg-100ng as a reference. Due to the different copy numbers of the target gene contained in templates of different species, gradient dilution of the template can be performed to determine the optimal template usage.