The QuantStudio 5 Real-Time PCR is a high-performance instrument equipped with a 384-well block. The system offers measurement of a broad range of genomic applications allowing use of probe-based assays and intercalating dyes.
qPCR is a method of measuring how much DNA in a sample contains a region (or regions) of interest. Like standard PCR, precisely-designed primers target specific regions that are (or may be) within the sample, and replicate that region over the course of 25-50 thermal cycles. Unlike standard PCR, the DNA is dyed with a fluorophore and the intensity of the fluorescence is measured at the end of each replication cycle. The cycle at which the fluorescence reaches measurable levels above background noise is referred to as the threshold cycle (CT). Control samples with known input quantities of DNA are run simultaneously, and their CT values are used to generate a standard curve so that the quantity of the experimental sample can be accurately calculated.
The image to the right shows a standard curve in triplicate. The cycle at which each sample’s curve intersects the horizontal line – the measurement threshold – is the CT value for that sample, and the standards’ CT values are evenly spaced to create a reliable equation for quantifying the experimental samples.
When only a single region is being targeted, the sample can be dyed with a generalized DNA fluorophore such as Sybr Green, which will bind to all dsDNA present; only one set of primers is used, so extraneous DNA isn’t amplified or measured. When multiple regions are of experimental interest, unique fluorescently labeled probes are added to the sample and fluoresce either when bound or when removed for DNA extension.
The QuantStudio 5 in the Genomics Facility is a 384-well instrument, enabling high-throughput sample processing. The Core only stocks reagents for quantifying Illumina sequencing libraries, but provides training on the QuantStudio so that researchers can use the instrument independently for a variety of needs. Please visit our iLab page to request training or reserve instrument time.
- Quantitative PCR analysis of next generation sequencing libraries
Please see our rates page for detailed pricing information.