RT-qPCR

Time:2022-3-20

RT qPCR: PCR is carried out with cDNA as template. In the process of PCR amplification, the PCR process is detected in real time by collecting fluorescent signals. Since there is a linear relationship between the CT value of the template and the initial copy number of the template in the exponential period of PCR amplification, it can be quantified

  • The amplification products of TA probe and target molecule are increased by using the specific hybridization sequence of TA probe and man probe; 
  • Non probes: including SYBR Green I or specially designed primers (such as lux primers) to indicate the increase of products through fluorescent dyes
  • In the process of real-time PCR amplification, fluorescence signals are collected and transformed into amplification and fusion curves. The specific data are baseline, fluorescence threshold and CT value. 
    Baseline: Generally speaking, the fluorescence value of cycle 3-15 is the baseline, which is caused by accidental error of measurement. 
    Threshold: the threshold is generally 10 times the standard deviation of the baseline. In practice, it can also be adjusted manually, just in the index period. 
    CT value: CT value is the number of PCR cycles when the fluorescence value reaches the threshold. So it is a parameter without unit. It is inversely proportional to the amount of initial template.

Steps:

  1. Configure PCR reaction solution: Takara rr820a Kit
    Prepare the following reaction system on ice.
    Reagent usage
    SYBR Premix Ex Taq II (2) 10.0μL
    PCR Forward Primer (10uM) 0.8μL
    PCR Reverse Primer (10uM) 0.8μL
    ROX Reference Dye II (50) 0.4μL
    CDNA solution 0.2 μ L
    DEPC water 6.0 μ L
    Total 20.0μL
  2. Two step amplification: Stage 1: pre denaturation 95 ℃ 30s
    Stage 2: PCR reaction: 95 ℃ 5S, 60 ℃ 30s, 40cycles
RT-qPCR

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RT-qPCR

error analysis