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Real-time PCR

Real time and Quantitative PCR

Introduction to Real-Time PCR

Real-time PCR is a type of quantitative PCR which measures the amount of cDNA or mRNA in a sample, either from a population of cells (tissue or cell culture), or recently even from a single cell. Real-time is used commonly to determine the expression of a gene's mRNA, and its expression levels (copy number of mRNA) during certain conditions (such as treating cells with a drug).  Real-time PCR can be used to compare normal (control) samples to disease samples, giving an idea as to expression changes which occur with pathogenesis.  Real-time PCR due to its sensitivity is also used in the detection of pathogens in the blood such as viruses.

The development of real-time quantitative PCR has eliminated the variability traditionally associated with quantitative PCR, thus allowing the
routine and reliable quantitation of PCR products.

Table of Contents

Introduction to Real-time PCR

Real Time PCR: Advantages of Real-time PCR

History of Real-Time PCR

Real-time PCR systems Available

Real-time PCR Protocols

Mechanism of Real-time PCR

Real Time PCR: Advantages of Real-time PCR

Each cell type expressess a set of mRNA molecules, known as a transcriptome.  In response to stimuli, cells are able to up-regulate or down-regulate factors such as protein enzymes inside the cell by regulating the mRNA levels of these factors.  mRNA levels are not always correlated with protein levels so some caution is necessary, however generally mRNA levels do loosely correspond to protein levels.  Measuring the mRNA levels of certain factors inside a cell using real-time pcr is a method which will give clues as to the amounts of these factors or proteins.

Traditionally, mRNA levels inside the cell were measured using Northern blotting.  This technique is regarded as a gold-standard in the measurement of gene expression/mRNA levels as it uses radiolabeled probe to label the RNA, which is then quantified using a scintillation counter giving very accurate readings. Northern blotting although very accurate had several disadvantages including the use of radioactivity. Northern blotting required the accurate measurement of mRNA and total RNA from cells extracted in order to compare samples. This was a problem because although detection methods were very accurate, error could be introduced into Northern blotting (or RNA dot/slot blotting) through differences in total RNA/mRNA levels between samples (ie cell treatments, different tissues, different animals, etc.).  It also required relatively large amounts of RNA which required large numbers of cells. Recently, mRNA gene quantification or real-time pcr methods have been improved and are easier to perform and do not require the use of radioactivity.  Researchers often only have access to small amounts of cells especially in the fields of stem cell research and primary cell research, and real-time pcr has made it possible to quantify mRNA levels from even very small numbers of cells and lately even single cells.  However, this extreme sensitivity of real-time pcr methods makes it vital to protect one's samples from contamination, which would lead to false results. Current real-time pcr methods and systems also do not require the measurement of mRNA or cDNA sample concentrations before real-time pcr is conducted.

History of Real-Time PCR

Higuchi et al.1,2 pioneered the analysis of PCR kinetics by constructing a system that detects PCR products as
they accumulate. This “real-time” system includes the intercalator ethidium bromide in each amplification reaction,
an adapted thermal cycler to irradiate the samples with ultraviolet light, and detection of the resulting fluorescence
with a computer-controlled cooled CCD camera. Amplification produces increasing amounts of double-stranded
DNA, which binds ethidium bromide, resulting in an increase in fluorescence. By plotting the increase in fluorescence
versus cycle number, the system produces amplification plots that provide a more complete picture of the
PCR process than assaying product accumulation after a fixed number of cycles.

External sites: see Real Time PCR at Real Time PCR Info!

References for Real-Time PCR

1. Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences.
Biotechnology 10:413–417.

2. Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR: Real time monitoring of DNA amplification reactions.
Biotechnology 11:1026–1030.

Real-Time PCR Protocols	Real-Time PCR Bioinformatics and Databases	Learn about Real-Time PCR	Real-Time PCR Kits and Products	Real-Time PCR Forum	Real-Time PCR Books