为了比较不同实时荧光定量 PCR 的扩增程序对荧光信号收集的影响, 优化出最佳方案, 并尽可能地成为普适性的实验方法, 以引物及探针为目标, 对其退火温度进行优化, 测试不同的RT-PCR扩增程序, 并以最终的△Fluorescence为依据判断程序优劣. 实验结果表明, 在设定探针的退火温度(62℃)之后,再给定一个较低的引物退火的温度(55℃)有利于荧光信号的收集, 并能得到更为标准的峰形图. 这说明实时荧光定量 PCR 的反应程序中, 设置双退火温度更有利于荧光信号的激发和收集, 更加便于实验结果的判读.%To investigate effects of different RT-qPCR amplification procedures on fluorescence signal collection, optimize the optimal scheme, and make it possible to be an experimental method of universality as far as possible, annealing temperature is optimized with primers and probes, and the different RT-PCR amplification programs are tested and the final delta is judged that it is based on fluorescence of the program. After setting the annealing temperature of the probe (62 ℃), the annealing temperature of a lower primer (55℃) is beneficial to the collection of fluorescence signals, and we will obtain more standard peak figure. The dual annealing temperature is set in the program of RT-qPCR, then it is propitious to excite and collect fluorescence signals, and is more convenient for the interpretation of experimental results.
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