Introduction to Real-Time PCR
Real-time PCR is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific nucleotide sequences in a sample. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real-time.
Two common methods for detection of products in real-time PCR are:
- Fluorescent dyes (e.g. SYBR green) that intercalate with any double-stranded DNA
- Sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target (e.g. TaqMan assays)
We recommend the use of SYBR green for detection. There are several advantages of using SYBR green. Multiple reactions can be set up rapidly and inexpensively with standard oligonucleotides. SYBR green will detect any PCR product, including nonspecific products and primer-dimers. Therefore, care must be taken during oligonucleotide design. Our primers are 20-mers with 55% G-C content and a single 3' -G or -C. Primer pairs are tested for specificity and absence of primer-dimer formation by PCR followed by gel electrophoresis.
Other detection systems such as molecular beacons and Invader and TaqMan assays are also routinely used at the facility. Although commercial kits are available, we use our own design, assembling our own reaction components. For details on real-time PCR protocols, see:
- Monitoring G-protein-coupled receptor signaling with DNA microarrays and real-time polymerase chain reaction. Methods Enzymol. 2002. Vol.345: 556-69. PMID: 11665639
- Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays. Nucleic Acids Res. 2002. Vol.30: e48. PMID: 12000853
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An overview of RT-PCR [PDF]
SYBR or TaqMan [PDF]