Real-time PCR

Quantitative 'real-time' PCR (qPCR) instruments measure the accumulation of signal generated by fluorescent dyes during each cycle of PCR. Real-time fluorescent signal can be generated using two distinct types of fluorescent detector chemistries:

dsDNA binding dyes, the most common of which is SYBR Green;
gene-specific probes labelled with fluorescent molecules

SCMB qPCR facility instruments can be calibrated to work with various fluorescent detectors. Please check the current instrument calibration status in the facility's RIMS documents, and contact us if you need custom calibration.

Facility staff are here to support you every step of the way. We can help with instrument selection and training, experiment design, setup, data analysis, and troubleshooting. We can provide users with labware, Master Mix, and clean reaction setup spaces for manual or robotic-assisted plate preparations.

Contact us

Contact Dr Vitaliya Sagulenko or SCMB Scientific Services for more information.

Applications

Equipment and location

SCMB RT-PCR facility is located in 76-346 and 76-348.

The following equipment is available:

QuanStudio 6 and Viia7 qPCR machines: 6-colours (green to far-red emission detection), 384-well plate block, QantStudio software
QIAGEN –Rotor-Gene-Q qPCR machine: 72-tubes rotor, 2 colours (green and yellow emission detection)
Eppendorf epMotion 5073 pipetting robotics system
Integra AssistPlus pipetting robot with Viafo and Voyager electronic 8- or 12-channel pipettes
Laminar-flow hood, multichannel pipets and plasticware for manual PCR setup
Qbit-3 fluorometer for fast and sensitive quantification of DNA, RNA, and Protein

Analysis Workstations in 76-111 provide access to the following software packages: PrimerExpress-3, Qaunt Studio, Rotor-Gene Q; Vialab for AssistPlus.

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.