Real-time Quantitative PCR

Cat. No. SER006

Service

Major Service Items

  • Detection System: LightCycler 1.5 (Roche)

  •  Primer & probe design

  • Quantitation assay (SYBR Green, Universal Probe Library & hybridization probe system)

  • Melting curve

  • PCR efficiency calibration

Our Experience

    Between Run: CV < 2%

    Within Run: CV < 1 %

    PCR Efficiency = 1.99 (As Figure)

Applications

  • Gene expression analysis, such as oncological or virological research parameters

  • Validation of DNA microarray result

  • Validation of gene knock-down result

  • Food pathogens & spoilage testing

  • Screening for genetically modified organisms (GMO)

Background information

The real-time PCR system is based on the detection and quantitation of a fluorescent reporter (Lee, 1993; Livak, 1995).  Advantages are:

  • Traditional PCR is measured at end-point (plateau), while real-time PCR collects data in the exponential growth phase

  • An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated

  • The cleaved probe provides a permanent record amplification of an amplicon

  • Requirement of 1000-fold less RNA than conventional assays

  •  No-post PCR processing (no electrophoretical separation of amplified DNA)

  •  Detection is capable down to a 2-fold change.

References

  •  Lee et al. (1993) Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res, 21, 3761-3766.

  •  Livak et al. (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl, 4, 357-362.