Basics of Real-Time PCR
by Jennifer Hornstein - 04/17/12
What is Real-Time PCR?
Real-Time PCR—also called quantitative polymerase chain reaction (qPCR)—is one of the most powerful and sensitive gene analysis techniques available and is used for a broad range of applications including quantitative gene expression analysis, genotyping, SNP analysis, pathogen detection, drug target validation and for measuring RNA interference. Frequently, real-time polymerase chain reaction is combined with reverse transcription to quantify messenger RNA (mRNA) and MicroRNA (miRNA) in cells or tissues.
For a more in depth explanation, watch the Life Technologies Real-Time PCR webinar series created for novice users; however, experienced users may also benefit from tips offered.
Real-Time PCR Steps
To perform Real-Time PCR, start with a basic PCR mix and add fluorescent labels to the PCR mix. A light source in the Real-Time PCR instrument then excites the fluorescence and a camera captures the fluorescent signals. As amplification proceeds, the fluorescence accumulation is captured by the instrument after every cycle and is translated into a Real-Time PCR graph.
With Real-Time PCR, There are Three Amplification Stages:
In the Exponential phase the reagents are in abundance and the PCR product doubles every cycle. In the Linear phase the reagents begin to run out and the PCR reaction slows down. In the Plateau phase the reagents are depleted and the PCR reaction stops.
Real-Time PCR focuses on the Exponential phase because it provides the most precise and accurate data for quantitation.
Within the Exponential Phase Two Values are Calculated:
- CT Value
The threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background. The PCR cycle at which the sample reaches this level is called the Cycle Threshold, Ct. The Ct value is used in downstream quantitation or prescence/absence detection.
Advantages of Real-Time PCR Over Traditional PCR
First, it is more sensitive which allows detection of low copy targets with greater confidence. It is also more quantitative which provides easy access to highly quantitative data. Real-Time PCR is faster. No gels are needed which saves time and effort. Because Real-Time PCR does not involve ethidium bromide or radioactivity it is much safer than traditional PCR.
Real-Time PCR Systems from Life Technologies use Two Main Fluorescence Detection Formats
- SYBR Green Dye
- TaqMan Probes
The SYBR fluorescence format uses a dye called SYBR Green, which binds non-specifically to double-stranded DNA. The DNA dye complex emits green light which is recorded by the Real-Time PCR instrument.
For SYBR Green detection it is important to run a Melting Curve analysis following Real-Time PCR to ensure that the desired amplicon was detected. The inflection point on the curve indicates the melting point of the amplicon. Contaminating DNA or primer dimers would show up as additional peaks separate from the desired amplicon peak.
The TaqMan probe fluorescence format uses two primers, a probe with a fluorescent reporter dye and a quencher, target DNA, and Polymerase.
The design of the probe is key. The Taqman probe is an oligonucleotide that contains a fluorescent reporter dye bound to the 5' end and a quencher on the 3' end. The probe is designed to bind to the target DNA sequence. While the dye and quencher are intact, there is no fluorescence. When the polymerase elongates, it is able to cleave the probe, separate the reporter from its quencher, and fluoresce. This fluorescent signal is captured by Real-Time PCR.
All Real-Time PCR formats detect fluorescence in real-time, and use the Ct value to perform quantitation and presence/absence amplicon detection.
Life Technologies offers a comprehensive set of Real-Time PCR products for routine and challenging applications such as real-time PCR-based gene expression, miRNA, copy number variation, and SNP genotyping analysis: from off-the-shelf gene-specific probe and primer sets, to everyday reagents and plastics, instrument systems, software, and everything in between.