Quantitative real-time RT-PCR (qRT-PCR) is widely and increasingly used in any kind of mRNA quantification, because of its high sensitivity, good reproducibility and wide dynamic quantification range. While qRT-PCR has a tremendous potential for analytical and quantitative applications, a comprehensive understanding of its underlying principles is important. Beside the classical RT-PCR parameters, e.g. primer design, RNA quality, RT and polymerase performances, the fidelity of the quantification process is highly dependent on a valid data analysis.

The software provided with real-time PCR instruments allows several types of data analysis:

1. normalisation of the raw data
2. measurement of the cycle number at which any increase in the fluorescence within each reaction vessel reaches significance
3. the data are used in conjunction with the results from internal or external standards to estimate the original number of template copies
4. melting curves are transformed to provide plots of –dF/dT against T (F = fluorescence and T= temperature) in which a peak (melting peak) occurs at the equilibrium temperature for each duplex

In general instrument specific software is easy to use and allows rapid and reproducible data analysis. In addition to the bundled software a range of third party utilities is available to improve the flexibility of real-time PCR data analyses.

from M.W. Pfaffl, J. Vandesompele and M. Kubista in Real-Time PCR: Current Technology and Applications

Further reading: Real-Time PCR

Labels: data analysis, PCR instruments, qPCR, qRT-PCR, quantitation, quantitative real time pcr

Unlike standard PCR, real-time PCR instruments measure the kinetics of product accumulation in each PCR reaction tube. Generally, no product is detected during the first few PCR cycles as the fluorescent signal is below the detection threshold of the instrument.

Most combinations of machine and fluorescence reporter are capable of detecting the accumulation of amplicons before the end of the exponential amplification phase. During this time the efficiency of PCR is often close to 100% giving a doubling of the quantity of product at each cycle. As product concentrations approach the nanogram per microlitre level the efficiency of amplification falls primarily because the amplicons re-associate during the annealing step. This leads to a phase during which the accumulation of product is approximately linear with a constant level of net synthesis at each cycle. Finally, a plateau is reached when net synthesis approximates zero.

Quantification in real-time PCR is done by measuring the number of cycles required for the fluorescent signal to reach a threshold level or the second derivative maximum of the fluorescence versus cycle curve. This cycle number is proportional to the number of copies of template in the sample. Real-time PCR quantification applications are discussed in detail in Bustin and Nolan (2009) Analysis of mRNA Expression by Real-Time PCR In: Real-Time PCR Logan, Edwards and Saunders, eds.; Wurmbach (2009) Validation of Array DataIn: Real-Time PCR Logan, Edwards and Saunders, eds.; and Wiseman (2009) Real-Time PCR: Application to Food Authenticity and Legislation In: Real-Time PCR Logan, Edwards and Saunders, eds.

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: fluorescence, PCR instruments, PCR machines, quantitation, real-time pcr

PCR machines with integrated fluorimeters and a mechanism for transferring excitation light from a source into the reaction vessel and then from the sample to a detector are required for real-time PCR. The heating blocks that are the mainstay of the standard PCR machine present several technical challenges in conversion to application in real-time PCR machines. The main problem being that the light must be channelled through the lid of the block and the cap of the reaction vessel across an air gap and then into the sample. Emitted light must then take the return path. Although blocks are used by several real-time PCR machines including the first commercial real-time PCR machine (Applied Biosystems 7700), the difficulties associated with them have led to the development of alternative designs for real-time pcr machines.

The LightCycler (LC24) was the forerunner of PCR machines that use air as the heating/cooling medium. Thermal transfer via air has the advantage of greater uniformity and rapidity than can be achieved on block-based PCR machines, besides allowing shortening of the light path. As well as differing in the choice of heating medium real-time PCR machines also provide a range of options for the light source and detection of fluorescence. Current, real-time PCR machines tend to allow the excitation and detection of multiple dyes so that internal standards and multiplex reactions are possible. There is also a tendency to build in a bias toward the use of either universal donor or universal recipient chemistry.

The cost of real-time PCR machines has fallen in tandem with continual improvement in their capability and accuracy. This has been the result of competition, the volume of sales and the introduction into the marketplace of improved designs dependent on new technology. These trends are unlikely to be reversed and will contribute to the growth in the popularity of real-time PCR. Real-time PCR machines are described and discussed in more detail in Real-Time PCR Machines (Logan and Edwards 2009. Chapter 2. Real-Time PCR: Current Technology and Applications. Caister Academic Press, Norfolk, UK.

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: fluorimeters, PCR instruments, PCR machines, real-time pcr, thermal cyclers

There are two general approaches used to obtain a fluorescent signal from the synthesis of product in Real-time PCR. The first depends upon the property of fluorescent dyes such as SYBR Green I to bind to double stranded DNA and undergo a conformational change that results in an increase in their fluorescence. The second approach is to use fluorescent resonance energy transfer (FRET). These methods use a variety of ways to alter the relative spatial arrangement of photon donor and acceptor molecules. These molecules are attached to probes, primers or the PCR product and are usually selected so that amplification of a specific DNA sequence brings about an increase in fluorescence at a particular wavelength.

A major advantage of the real-time PCR instruments and signal transduction systems currently available is that it is possible to characterise the PCR amplicon in situ on the machine. This is done by analysis of the melting temperature and/or probe hybridisation characteristics of the amplicon within the PCR reaction mixture. In the intercalating dye system, the melting temperature of the amplicon can be estimated by measuring the level of fluorescence emitted by the dye as the temperature is increased from below to above the expected melting temperature. The methods that rely upon probe hybridisation to produce a fluorescent signal are generally less liable to produce false positive results than alternative methods such as the use of intercalating dyes to detect net synthesis of double stranded DNA (dsDNA) followed by melting analysis of the product.

Hybridisation, ResonSense and hydrolysis probe systems give fluorescent signals that are only produced when the target sequence is amplified and are unlikely to give false positive results. An additional feature of the hybridisation, ResonSense and related methods is the possibility to measure the temperature at which the probes disassociate from their complementary sequences. This measurement gives a further verification of the specificity of the amplification reaction. An important feature of many of the probe systems is their compatibility with multiplexing due to the availability of fluorophores with resolvable emission spectra.

from N.A. Saunders in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: fluorescence, fluorescent dyes, hybridisation, multiplexing, PCR instruments, real-time pcr, ResonSense, signal transduction