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Real-Time PCR (qPCR)



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Real-Time PCR (qPCR)

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Real-time quantitative PCR, also known as qPCR, combines the PCR amplification and detection into a single step, thus eliminating the need to detect products using gel electrophoresis and enabling the PCR method to be truly quantitative. Real-time PCR instrument systems utilize fluorescent dyes to detect the accumulation of PCR products during the exponential phase of the reaction, which allows for fast and precise product quantification and objective data analysis.

Real-time PCR instruments are used to perform the PCR as well as the real-time detection and monitoring of the resulting fluorescent signal. The Applied Biosystems family of qPCR systems, combined with our gold standard qPCR master mixes and TaqMan® probe-based assays for gene expression and genetic variation, make qPCR more accessible than ever. These systems offer cutting-edge software designed to support your application, and are flexible to accommodate the real-time chemistry of your choice.

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Overview of real-time PCR/qPCR
Real-time polymerase chain reaction (PCR) is one of the most powerful and sensitive gene analysis techniques. It is used for a broad range of applications including quantitative gene expression analysis, SNP genotyping, copy number analysis, pathogen detection, drug target validation, and RNA interference analysis. The substrate or input for real-time PCR is DNA, thus for analysis of RNA species, such as messenger RNA (mRNA), microRNA (miRNA), or other noncoding RNA, samples are reverse transcribed to convert RNA into cDNA prior to real-time PCR (this is called real-time RT-PCR).

Real-time PCR defined
Real-time PCR is a type of polymerase chain reaction (PCR) in which data are captured at every step of thermal cycling (rather than at the end, as in traditional or end-point PCR). (See Real-Time PCR vs. Traditional PCR for a comparison.) Like all PCR strategies, reactions contain a large molar excess of oligonucleotide primers that are complimentary to the 5’ and 3’ ends of the desired target sequence or amplicon. These PCR primers “prime” synthesis of one copy of the amplicon at each round of thermal cycling. Reaction products are fluorescently labeled using one of two main strategies (See TaqMan® Assay and SYBR® Green Chemistries for more information): SYBR® Green I dye—a fluorescent dye that specifically binds double-stranded DNA TaqMan® fluorogenic probes—target-specific oligonucleotides with a fluorescent dye on one end and a nonfluorescent quencher on the other end. TaqMan® probe fluorescence is quenched when the probe is intact, but when it is destroyed by the DNA polymerase during PCR, the fluorescent probe is physically separated from the quencher and produces signal. Both fluorescent labeling strategies result in accumulation of fluorescent signal in proportion to the quantity of PCR reaction products. (See a video of how real-time PCR works.)

Fast real-time PCR
Fast real-time PCR, as it name implies, simply uses a faster thermal cycling protocol than standard real-time PCR. To get the most benefit from fast thermal cycling and the shortest reaction times, without compromising your data, you’ll need a real-time PCR master mix and instrument optimized for Fast cycling. See Fast real-time PCR solutions from Applied Biosystems and Invitrogen for product information.

Real-time PCR is qPCR (quantitative PCR)
Real-time PCR is often referred to as qPCR because it can be used to quantify the amount of target DNA (or cDNA) in the samples prior to thermal cycling. Quantitation is based on the cycle threshold (Ct): During the exponential phase of amplification, target quantities double at each PCR cycle. The Ct is the PCR cycle at which fluorescence from reaction products becomes distiguishable above background; this occurs at the beginning of the exponential phase of the PCR. The lower the Ct, the more abundant the target was in the original sample.
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