Quantitative "real time" PCR (qPCR) measures the accumulation of fluorescent DNA amplification products as they are generated during each cycling step of a polymerase chain reaction.

This produces a "real-time" plot of fluorescence versus cycle number, where the fluorescence is proportional to the amount of amplified product.

Quantitative data are obtained by measuring the cycle threshold (Ct) - the cycle number at which fluorescent products are first detected, rather than the final amount of fluorescent product accumulated after a fixed number of cycles as in standard PCR.

The higher the amount of initial nucleic acid template in a reaction, the sooner fluorescence is detected and the lower the Ct value that is measured.

This enables us to quantify the relative abundance of various DNA target sequences in a sample, or absolute abundance of a target by comparison with appropriate standards.

The kinetic data produced during the reaction are useful for quality control without having to open reaction tubes and analyse their contents.

For example, the PCR amplification efficiency can be determined during the initial (exponential) phase of the reaction, and frequently approaches the theoretical maximum of 2n when amplifying small DNA target sequences.

Fluorescent "melt curves" obtained by heating the final product can determine if the expected PCR amplicon is contaminated by non-specific amplification products.

Contact us

Contact Dr Michael Nefedov for more information.

Applications

Comprehensive service:

  • locate suitable sequences as a target for your PCR
  • design oligos for your relative or absolute quantitation using SYBR green or TaqMan assays.
  • test multiple housekeeping genes for your Internal control
  • propose best strategy for real-time PCR assays.

Using most recent advances in nucleic acids isolations technologies:

  • isolate DNA or RNA/miRNA free of contaminants and cross contamination suitable for real-time PCR and NGS
  • convert RNA to cDNA via Reverse Transcription with Oligo d(T)18 or random hexamer
  • assemble and run real-time PCR reactions in 384-well format (multiple plates)
  • analyse data, produce statistical and graphic reports in various formats.

Equipment and training

Equipment

Our instruments enable you to monitor fluorescence during the PCR.

We offer discounted PCR plates or tubes, SYBR Green and Taqman (ABI).

Our equipment includes:

  • ABI-7900HT, 384-well plate block and low-density arrays attachment (micro fluidic card)
  • ABI-ViiA7, 384-well plate block with high resolution melting analysis
  • Eppendorf epMotion 5075 Robotics System (available for setting up assays for real-time PCR)
  • 384well Gradient Agilent SureCycler8800 
  • Corbett Rotorgene 6000 -36 & 72 tube rotors
  • Corbett CAS 1200 Robotics System (available for setting up assays for real-time PCR)
  • Maxwell16 DNA/RNA/Protein isolation robot (various samples types accepted).

You can use our clean room, hood, multichannel pipettes, filter tips, plasticware and SYBR and TAQMAN reagents to set up the PCR assays.

We also offer:

  • four PCs with software
  • Primer Express for primer/probe design
  • SDS (sequence detection software) for analysis of data from ABI7900 runs
  • ViiA7 RUO software for analysis of data from ViiA7 runs, with a license for your own PC.

User guides and download instructions

Visit our online Science Booking Calendar (UQ login required) for more information about our equipment and software, including manufacturers' user guides and download instructions.

Select the relevant piece of PCR equipment from our School's equipment menu.

Contact us for login details if you're an external academic.

Training

We can show you how to apply the available technology to your requirements and advise you on available chemistries and reagents/consumables, primer and probe design, optimisation of assays and quantitation methods.

We also provide training for instrument use and data analysis.

Watch our video

Our instructional video demonstrates how to use the simpler aspects of our RT-PCR facilities.

Detection chemistries

Several alternative fluorescence chemistries have been devised to detect DNA molecules produced during PCR.

You can use all of them with our instruments.

The two most frequently used are:

  • SYBR Green dye: this binds to the minor groove of double-stranded DNA, and the SYBR-dsDNA complex emits green fluorescence when excited with light of lower wavelength. This chemistry is relatively cheap, but isn't specific and produces fluorescence from all dsDNA amplified including non-target sequences and primer dimers if present
  • TaqMan fluorogenic probes: named after the computer game PacMan, this uses a single-stranded DNA probe of ~15-30 nucleotides (labelled with a fluorogenic dye at the 5' end and a fluorescence quencher at the 3' end) homologous to a target sequence between the PCR priming sites.

View an image of our fluorescent dyes spectra.

During the extension phase of PCR, the Taq DNA polymerase encounters the bound probe and cleaves it, hence releasing the detection dye from the effect of the quencher. This results in enhanced fluorescence when the dye is excited with incident light.

The extra specificity conferred by the detection probe has advantages for many applications, for example diagnostic applications where false positives are a problem.

Data analysis

The Ct value for an assay is related to the abundance of template in that reaction.

For the absolute quantitation method, a standard curve of a plot of Ct value against the log of the template concentration allows template concentration for unknown samples to be determined.

For the relative quantitation method, which is frequently used for mRNA analysis, the abundance of a transcript of a gene of interest is measured relative to that of an endogenously expressed gene such as actin or rRNA (housekeeping gene).

Relative quantitation works best when the genes being compared are amplified with similar PCR efficiencies, but corrections can be made if measured differences in efficiency are known. We can advise you on appropriate experimental design and data analysis strategies, and how to set up spreadsheets to process data.

    Sample and set-up requirements

    Users are responsible for obtaining suitably purified and diluted genomic DNA or cDNA samples and setting up their own reaction tubes.

    We can advise you on how best to do this, but generally you'll need:

    • DNA or cDNA samples free of "Taq inhibitor", typically ~20 ng cDNA / 5 μL
    • primer solutions, typically 1uM but requires optimisation.
    • TaqMan probe (if required), typically 2uM but requires optimisation.
    • master mix kit containing a suitable Taq DNA polymerase and buffer.

    For consistent results from run to run, we recommend you choose a commercial kit rather than prepare your own, despite this representing the largest single cost for accessing this technology.

    Contact us for more details, and for details about reaction tubes/strips/plates.

    Bookings, charges and considerations

    Charges

    Considerations

    UQ users have priority of access.

    You'll need to undertake the relevant building and other inductions before using equipment. 

    Bookings

    Internal users

    Visit our online Science Booking Calendar (UQ log-in required) for more information about our equipment, including manufacturers' user guides.

    Select the piece of PCR equipment you want to book from our School's equipment menu and follow the booking instructions.

    Contact the Facility Manager for a Request for Internal Services form.

    External users

    Contact the Facility Manager to arrange:

    • access to our booking system
    • keycard access to our facilities (you'll need to fill in an authorisation form)
    • raising a purchase order.