TaqMan One Step RT-qPCR kit first uses reverse transcriptase to reverse transcribe RNA into cDNA, and then uses cDNA as a template for continuous PCR amplification in a single-tube closed tube reaction system by heat-activated DNA amplification enzyme, and uses fluorescent labeled Probe for hydrolysis and luminescence. The luminous signal was detected.

RT-PCR

First, reverse transcriptase is used to synthesize cDNA using RNA as template, and then the synthesized cDNA is used as template. Through the three steps of thermal denaturation, primer annealing and chain extension of PCR amplification, a large number of DNA fragments can be amplified in a short time.

Fluorescence Detection

The TaqMan probe method uses a TaqMan probe with a fluorescent substance at the 5 'end and a quenched substance at the 3' end for fluorescence detection. When the probe is not decomposed by Taq enzyme, the 5 'end fluorescent material is restricted by the 3' end quenched material and cannot fluoresce. When the TaqMan probe is decomposed, the fluorescent material at the 5 'end will be free and emit fluorescence. During the annealing process after the fluorescent probe is added to the PCR reaction solution, the fluorescent probe will bind to the template pairing region. During the extension of the PCR reaction, the 5'3' exonuclide activity of Taq DNA polymerase breaks down the fluorescent probe hybridized with the template, and the free fluorescent substance fluoresces. By detecting the fluorescence intensity in the reaction system, the purpose of detecting the amplification of PCR products can be achieved.

1.  PCR Procedure (two-step method)

• Reverse transcription: 50°C 10 minutes
• Variability: 95°C 2 minutes
• Variability: 95°C 10~20 seconds
• Annealing/Extension: 60°C 20~60 seconds

2. PCR Procedure (three-step method)

• Reverse transcription: 50°C 10 minutes
• Variability: 95°C 2 minutes
• Variability: 95°C 10~20 seconds
• Annealing: 56-~64°C 10~360 seconds
• Extension: 72°C 10~60 seconds