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:
- 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 - Two step amplification: Stage 1: pre denaturation 95 ℃ 30s
Stage 2: PCR reaction: 95 ℃ 5S, 60 ℃ 30s, 40cycles
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error analysis