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Real-Time (Quantitative) PCR
- Two StepOnePlus and a QuantStudio3 Real Time PCR Systems
- Real time fluorescence monitoring of PCR products
- Absolute quantitation
- Relative gene expression
- Fast qPCR (<40 minute run time)
- Two types of chemistry can be used:
- SYBR Green - Double-stranded DNA-binding dye allows measurement of the accumulation of PCR products
- TaqMan - Fluorogenic probes for detection of specific PCR products, allowing allelic discrimination.
- Machines can also be used for high-resolution melting studies
What is Read-Time PCR?
- It is the ability to monitor the accumulation of fluorescent products in real time throughout the PCR procedure
- Reactions are characterised by the cycle number (Ct) at which products are first detected, rather than the amount of target accumulated after a fixed number of cycles
- The more of a specific target nucleic acid is present in a sample, the sooner a significant increase is detected and the lower the Ct value which is measured
- Absolute or relative quantitation of nucleic acids can be achieved over many orders of magnitude