Multiplex Real-Time PCR can be applied to relative quantification experiments where the gene of interest and reference gene are co-amplified in the same reaction. Multiplex Real-Time PCR can also be used for allelic discrimination assays, where two differentially labeled probes [...]
Developing multiplex Real-Time PCR assays can be difficult and time-consuming. As the reaction complexity increases, significant optimization may be required to generate reliable data. It can be a challenge to develop multiplex assays that amplify all targets with equal efficiency. [...]
Multiplex Real-Time PCR is a technique in which multiple target sequences are amplified and detected in a single PCR reaction. Amplified sequences are distinguished by the use of different dyes conjugated to the probes. The number of targets that can [...]
The specificity of any Real-Time PCR assay, whether TaqMan probe or SYBR Green I, is determined by the quality of the assay design. Non-specific amplification can occur for both SYBR Green I or TaqMan probe methods if the assay design [...]
There are two major detection chemistries used for Real-Time PCR: enzymatic and hydrolysis (TaqMan) probe-based chemistries, and DNA-binding SYBR Green I dye-based chemistry. Additional detection chemistries include Molecular Beacons, Scorpions probes and LUX primers.
I am currently running traditional PCR; can I use the same template, primers, and reagents to run Real-Time PCR?
In some cases it is possible to convert existing traditional PCR assays into Real-Time PCR assays, with a few considerations around primer design and master mix. Primer design is one of the first considerations for converting a traditional PCR assay. [...]
There are three phases of PCR amplification: exponential, linear, and plateau. The exponential phase is the first phase of PCR amplification. Reaction components are in excess, there is an exact doubling of product each cycle, and the reaction is specific [...]
Some basic Real-Time PCR terms and their definitions are: Amplification plot – Plot of fluorescent signal versus cycle number. Baseline – The initial cycles of PCR where there is little to no change in fluorescence. Threshold – The arbitrary level [...]
Real-Time PCR has been used in quantification of gene expression, viral quantification, validation of array data, pathogen detection, and allelic discrimination.
One advantage of Real-Time PCR over traditional PCR is that it is a closed-tube system requiring no post-PCR processing. Real-Time PCR has higher precision, increased sensitivity (down to one copy), increased dynamic range (greater than 8 logs), and high resolution [...]