Real-Time PCR

Real-Time Quantitative PCR or qPCR is currently the gold standard for accurate and sensitive quantification of specific nucleic acid sequences in biological samples.  It combines fluorescent based assays with the conventional polymerase chain reaction to simultaneously amplify and detect accumulation of PCR product in 'real- time'.  Changes in fluorescence measured at initial amplification are directly proportional to the input amount of target DNA or RNA and this is the basis of subsequent quantitative analysis.

Application

Real-Time PCR has a wide range of applications across diverse areas of scientific research, diagnostics and biotechnology. Common applications employ relative quantitative methods such as in gene expression profiling, microRNA and siRNA analysis. Other methods require absolute quantification such as determination of gene copy number or viral load and library validation for sequencing. By combining real-time assays with post-run reads, this technology can also be used for allelic discrimination or SNP detection and high resolution melt curve analysis.

Instrumentation:

The core facility operates two Real-Time PCR instrument platforms:

The ABI7900HT Sequence Detection System is a high throughput Real-Time PCR instrument that detects and quantitates nucleic acid sequences. This instrument is optimised for use with Taqman and Sybr Green chemistries and offers:

  • Interchangeable thermal cycling block capability to allow flexibility of sample set up in 96 or 384 well plate formats and Micro Fluidic Card arrays.
  • Continuous wavelength detection to support the use of multiple fluorophores in a single reaction.
  • Sequence Detection Software to provide tools for instrument operation, data collection and primary analysis.
  • RQ Manager Software to proceed to high throughput secondary data analysis.
  • Primer Express software for assay design requirements.

The Roche LightCycler 480 System is a medium to high throughput amplification and quantification instrument. It offers many of the above features such as flexibility of plate format and versatility of mono- or multicolour channels. The LightCycler 480 software uses unique Roche algorithms for analysis of a range of applications. This instrument is ideal for Sybr Green screening and is also optimised for use with Roche Real-Time Ready assays based on their Universal Probe Libraries. Researchers should note that only Roche reagents & consumables may be used on this this instrument.

Expertise & Services:

The Real-Time PCR core facility provides a range of technical services to researchers from initial experimental design to results analysis. Users of the facility are encouraged to become independent users and training is provided on sample set-up, instrument operation, data interpretation & QC and results analysis. Once training is complete, continued technical support is available as required.

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