Real-Time Polymerase Chain Reaction
In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene expression as a result of physiology, pathophysiology, or development. This method can be applied to model systems to measure responses to experimental stimuli and to gain insight into potential changes in protein level and function. Thus physiology can be correlated with molecular events to gain a better understanding of biological processes. For clinical molecular diagnostics, real-time PCR can be used to measure viral or bacterial loads or evaluate cancer status. Here, we discuss the basic concepts, chemistries, and instrumentation of real-time PCR and include present applications and future perspectives for this technology in biomedical sciences and in life science education.
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction ((Q-PCR/qRT-PCR)or kinetic polymerase chain reaction (KPCR), is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, Real Time-PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes.
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. This is a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) 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.
Advantages and Limitations of Real- Time PCR
There are many methods in molecular biology for measuring quantities of target nucleic acid sequences. However, most of these methods exhibit one or more of the following shortcomings: they are time consuming, labor intensive, insufficiently sensitive, nonquantitative, require the use of radioactivity, or have a substantial probability of cross contamination. These methods include but are not limited to Northern and Southern hybridizations, HPLC, scintillation proximity assay, PCR-ELISA, RNase protection assay, in situ hybridization, and various gel electrophoresis PCR end-point systems.
Real-time PCR has distinct advantages over these earlier methods for several reasons. Perhaps the most important is its ability to quantify nucleic acids over an extraordinarily wide dynamic range (at least 5 log units). This is coupled to extreme sensitivity, allowing the detection of less than five copies (perhaps only one copy in some cases) of a target sequence, making it possible to analyze small samples like clinical biopsies or miniscule lysates from laser capture microdissection. With appropriate internal standards and calculations, mean variation coefficients are 1-2%, allowing reproducible analysis of subtle gene expression changes even at low levels of expression. In addition, all real-time platforms are relatively quick, with some affording high-throughput automation. Finally, real-time PCR is performed in a closed reaction vessel that requires no post-PCR manipulations, thereby minimizing the chances for cross contamination in the laboratory.
However, there are several limitations to real-time PCR methods. The majority of these are present in all PCR or RT-PCR-based techniques. Real-time PCR is susceptible to PCR inhibition by compounds present in certain biological samples. For example, clinical and forensic uses for real-time PCR may be affected by inhibitors found in certain body fluids such as hemoglobin or urea. Food microbiological applications may encounter organic and phenolic inhibitors. To circumvent this problem, alternative DNA polymerases (e.g., Tfl, Pwo, Tth, etc.) that are resistant to particular inhibitors can be used. Other limitations primarily concern real-time PCR-based analysis of gene expression. Because of the necessary use of RNA in an extra enzymatic step, more problems have the opportunity to occur. RNA itself is extremely labile compared with DNA, and therefore isolation must be carefully performed to ensure both the integrity of the RNA itself and the removal of contaminating nucleases, genomic DNA, and RT or PCR inhibitors. This can be a problem with any sample source, but clinical samples are of special concern because inconsistencies in sample size, collection, storage, and transport can lead to a variable quality of RNA templates. Conversion of RNA to cDNA during the RT reaction is also subject to variability because multiple reverse transcriptase enzymes with different characteristics exist, and different classes of oligonucleotides (e.g., random, poly-dT, or gene-specific primers) can be used to prime RT.
Probably the largest present limitation of real-time PCR, however, is not inherent in the technology but rather resides in human error: improper assay development, incorrect data analysis, or unwarranted conclusions. In our experience using real-time PCR for gene expression analysis, real-time PCR primer sets must be designed and validated by stringent criteria to ensure specificity and accuracy of the results. For microbiology, false positives or negatives must be considered when designing an assay to detect pathogens. Amplification and melting curves must be visually inspected while independent calculations based on these curves should be double-checked for accuracy. Real-time PCR gene expression analysis measures mRNA levels and, therefore, only suggests possible changes in protein levels or function rather than demonstrating them. And although there is a tight connection between gene expression and gene product function, this is certainly not always the case, and formal demonstration may be needed for a given research project. Of course, conclusions based on data derived from real-time PCR are best utilized when the biological context is well understood.
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