Quantitative PCR (QPCR) or real-time RT-PCR is a sensitive, reproducible and accurate method for determining the DNA or mRNA levels in tissues or cells. The amount of PCR product produced in every cycle step of the PCR reaction is monitored, which may be accomplished directly with intercalating dyes which bind to the double stranded DNA (e.g. SYBR Green); or indirectly with fluorescent probes (e.g. TaqMan or Universal ProbeLibrary, UPL). In a conventional PCR reaction, the PCR product amount is measured as an end-point analysis on an agarose gel. In a QPCR reaction, the key feature is the ability to monitor the DNA amplification process as it happens, in “real-time.” QPCR delivers powerful capabilities for generating and analyzing data for a wide variety of applications.
The Feinstein Institute’s QPCR Core Facility, located on the first floor of the North Wing in Rooms 1261 and 1263. houses 3 machines: one Applied Biosystems (ABI) 7900HT Real-Time PCR System with Sequence Detection System (SDS) software and two Roche Applied Science LightCycler480 PCR Systems with software. Each system is equipped to perform QPCR in a 96 or 384-well format. The ABI 7900HT is also equipped with a TaqMan Low Density Array (TLDA) block, for use with pre-loaded TaqMan Gene Expression Assays. The TaqMan Array Card is a 384-well micro fluidic card that enables a researcher to perform 384 simultaneous reactions without the need to use liquid-handling robots or multichannel pipettes to fill the card. This micro fluidic card allows for 1-8 samples to be run in parallel against 12-384 TaqMan Gene Expression Assay targets that are pre-loaded into each of the wells on the card. Researchers are able to use Applied Biosystems premade TLDA Gene Signature Panels (for Angiogenetics, Apoptosis, Alzheimer’s, Immune etc.) or design their own custom TLDA cards.
All three instruments can be used for Absolute or Relative Quantitative gene expression analysis of numerous sample types, SNP’s and allelic discrimination assays, Plus/Minus assays and dissociation curve melting point analysis. All these are achieved through the Real-Time or End Point spectrofluorogenic detection of PCR over user programmable thermo cycling conditions. Kits and reagents from various vendors are available for both SYBR Green and Probe Based chemistries. The Facility fully stocks the complete Universal ProbeLibrary Set (#1 – #165). Additionally, various human, rat, and mouse Reference Genes are available.
The Facility provides real-time PCR chemistries, consultation, experimental design consultation, assistance in data analysis and troubleshooting. Machines are available 24 hrs per day. The facility does not setup reactions for investigators, but support is available upon request. Machine operations are self served for experienced users. The requirement is that the user be fully trained, can run a plate without assistance, and is able to take care of general instrument needs. Training sessions are highly recommended and can be provided for first time users. An online calendar to reserve time for the machine is available to all registered users. Please notify the manager and make a request to join the calendar.
Please note that the protocols, instructions, and recipes provided by the Core are not recommendations; they are just examples of how you can run your samples. The recipes work in most cases but not all, and it is your responsibility as a researcher to ultimately select your assay design, optimize, and decide how to run your samples/project.
For further information or if you have questions regarding the operation of the facility or other technical questions, please contact the manager Valerie Boone at firstname.lastname@example.org, or at (516) 562-1096.