Real-Time PCR

Fluorescence-based real-time PCR is an established method for precise, efficient and rapid nucleic acid detection. It combines amplification and detection in a single step. The sensitivity of this method permits reliable detection of small amounts of starting template. Because the PCR product is detected using fluorescent dyes in real time, it is quantitative.   

Real-Time PCR Key Applications:

  • Gene expression analysis
  • microRNA and non-coding RNA analysis
  • SNP analysis
  • Genotyping/allelic discrimination

Core Real-Time PCR Instruments:

The Research Core Facility has an Applied Biosystems 7900HT Fast Real-Time PCR System and three Bio-Rad CFX96 Fast Real-Time PCR Systems.

The ABI 7900HT is the only real-time quantitative PCR system that combines 96- and 384-well plate compatibility and the TaqMan® Low Density Array. With optional fast real-time PCR capability, this system reduces run time to about 35 minutes in a standard 96-well format, or about 55 minutes in a 384-well plate. Key applications include gene expression quantitation and the detection of single nucleotide polymorphisms (SNPs) using the fluorogenic 5' nuclease assay. To induce fluorescence, the 7900HT system distributes light from an argon laser excitation source to all sample wells via a dual-axis synchronous scanning head. It then directs the resulting fluorescent emission through a spectrograph to a CCD camera. Emission wavelengths from 500-660 nm are monitored allowing the simultaneous detection of multiple fluorophores. The system is compatible with FAM/SYBR Green I, VIC/JOE, NED/TAMRA/ Cy3, ROX/Texas Red and Cy5 fluorescent dyes.

The CFX96 is a six-channel real-time PCR system that combines advanced optical technology with precise thermal control to deliver sensitive, reliable detection. The system’s solid-state optical technology (six filtered LEDs, each with a corresponding filtered photodiode) maximizes fluorescence detection for specific dyes in specific channels, providing sensitive detection for quantitation and target discrimination. Data is collected from all wells during data acquisition. At every position and with every scan, the optics shuttle is reproducibly centered above each well, so the light path is always optimal and there is no need to sacrifice data collection on one of the channels to normalize to a passive reference. Users can select multiple data acquisition modes, including a one-color fast scan for SYBR green. Thermal gradient features can be used to optimize reactions in a single run. The new CFX Manager software has advanced analysis tools for performing normalized gene expression.