Protocols for Real-Time Quantitative PCR
GUIDELINE
The real-time quantitative PCR (qRT-PCR) technique refers to the addition of fluorescent groups to the PCR reaction system. The whole PCR process is monitored in real-time using fluorescence signal accumulation, and finally, the unknown template is quantified by a standard curve. Currently, the fluorescent chemical components used in real-time quantitative PCR can be generally classified into two types, fluorescent probes, and fluorescent dyes.
METHODS
- Primers were designed according to the mRNA of specific genes and sent to the primer synthesis company for synthesis.
- Use an RNA extraction kit and extract RNA.
- Using the kit, synthesize cDNA synthesis.
- Determine the contents of the mount and arrange the order of sample discharge on the record.
- Prepare the qPCR reaction system according to the below table.
| Ingredients | Single dose | Final concentration |
| Realtime PCR Master Mix | 7.5 mL | 1× |
| Primer mixture (10 mM) | 0.6 mL | 0.4 mM |
| cDNA template | X mL | No more than 10% of the total reaction system |
| H2O | Add up to 15 mL |
- Set up cycles. 95°C for 2 min; 95°C for 10 s, 55°C for 15 s, 72°C for 10 s, 40 cycles; 95°C for 1 min, 60°C for 30 s, 95°C for 30 s.
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NOTES
In the experiment, the addition of a sample was arranged in the same row as far as possible. In addition, the three replicates in the formal experiment cannot be arranged on the same plate, and the experiments in the three replicates need to be separated, but the experiments in the same replicate cannot be separated into two plates.
