FAQ: How do I use qPCR to determine the concentration of my material?

Quantitative PCR (qPCR) uses real-time fluorescence to measure the quantity of DNA present at each cycle during a PCR. A wide variety of approaches have been developed for generating a fluorescent signal, the most common of which use either hydrolysis probes (e.g., TaqMan®), or a double-stranded DNA binding dye, (e.g., SYBR® Green). At a point where the qPCR fluorescence signal is detectable over the background fluorescence, a quantification cycle, or Cq value, can be determined. Cq values can be used to evaluate relative target abundance between two or more samples. Alternatively, they can be used to calculate absolute target quantities in reference to an appropriate standard curve, derived from a series of known DNA dilutions. Typically, qPCR instrument software will perform the necessary plotting and calculations for concentration determinations. Alternatively, the NEB online tools include a qPCR calculator, which can be found at NEBiocalculator.neb.com.