We use real-time PCR to amplify and simultaneously quantify a targeted DNA/RNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific nucleotide sequences in a sample. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real-time.
Our CoRE facility is equipped with two ABI PRISM 7900HT sequence detection systems, configured to use 384-well or 96-well plates with SYBR green or TaqMan probes and capable of unattended 24-hour operation using a robotic arm to process plates in the queue. The facility has a QX100 Droplet Digital PCR System. It has many potential applications, including the detection and quantification of low levels of pathogens, rare genetic sequences, copy number variations, relative gene expression in single cells, and analysis of heterogeneous DNA methylation. The facility provides access to the Nanostring Technologies nCounter platform that allows for highly multiplexed (up to 800 targets per assay) digital quantification of nucleic acids. An eight channel 384/96-well format Beckman pipetting robot (Beckman Biomek NXp Laboratory Automation Workstation) is available for setting up PCR reactions in 384 and 96 well format. A Qiagen BioRobot Universal system is available for automated large-scale RNA and DNA isolation.