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. qPCR is a routinely used method within mutation detection, genotyping, and molecular diagnostics assays and is commonly used for measuring gene expression and copy number variation.
qPCR can also be modified to detect and quantitate RNA by adding a reverse transcriptase step upstream of the qPCR assay to generate cDNA (i.e., RT-qPCR). Reverse transcription can be performed separately from qPCR or directly in the qPCR mix (i.e., one-step RT-qPCR). One-step workflows are commonly favored in molecular diagnostic assays and where sample inputs may be limiting. Separate cDNA synthesis followed by qPCR (i.e., two-step RT-qPCR) is preferred when multiple interrogations will be made of the same starting material or where archiving of cDNA may be required.
TaqMan® is a registered trademark of Roche Molecular Systems, Inc.
SYBR® is a registered trademark of Molecular Probes, Inc.
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