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