Real-time polymerase chain reaction
Encyclopedia
In molecular biology
, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/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 oligonucleotide
s that are labeled with a fluorescent
reporter which permits detection only after hybridization of the probe with its complementary DNA target.
Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA
and Non-coding RNA
in cells or tissues.
Abbreviations used for real-time PCR methods vary widely and include: RTQ-PCR, Q-PCR or qPCR. Real-time reverse-transcription PCR is often denoted as: qRT-PCR, RRT-PCR, or RT-rt PCR. The acronym "RT-PCR" commonly denotes reverse-transcription PCR and not real-time PCR, but not all authors adhere to this convention.
Cells in all organisms regulate gene expression
and turnover of gene transcripts (messenger RNA
, abbreviated to mRNA), and the number of copies of an mRNA transcript of a gene in a cell or tissue is determined by the rates of its expression and degradation.
Older methods were used to measure mRNA abundance: Differential display
, RNAse protection assay and Northern blot
. Northern blotting
is often used to estimate the expression level of a gene by visualizing the abundance of its mRNA transcript in a sample. In this method, purified RNA is separated by agarose gel electrophoresis, transferred to a solid matrix (such as a nylon membrane), and probed with a specific DNA or RNA probe
that is complementary
to the gene of interest. Although this technique is still used to assess gene expression, it requires relatively large amounts of RNA and provides only qualitative or semiquantitative information of mRNA levels.
In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction
is a common method for amplifying DNA; for mRNA-based PCR the RNA sample is first reverse transcribed to cDNA
with reverse transcriptase
.
Development of PCR technologies based on reverse transcription
and fluorophore
s permits measurement of DNA amplification during PCR in real time, i.e., the amplified product is measured at each PCR cycle. The data thus generated can be analysed by computer software to calculate relative gene expression in several samples, or mRNA copy number. Real-time PCR can also be applied to the detection and quantification of DNA in samples to determine the presence and abundance of a particular DNA sequence in these samples.
in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNA dyes such as SYBR Green
will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer
). This can potentially interfere with, or prevent, accurate quantification of the intended target sequence.
Like other real-time PCR methods, the values obtained do not have absolute units associated with them (i.e., mRNA copies/cell). As described above, a comparison of a measured DNA/RNA sample to a standard dilution will only give a fraction or ratio of the sample relative to the standard, allowing only relative comparisons between different tissues or experimental conditions. To ensure accuracy in the quantification, it is usually necessary to normalize expression of a target gene to a stably expressed gene (see below). This can correct possible differences in RNA quantity or quality across experimental samples.
Fluorescent
reporter probes detect only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fluorescent probes can be used in multiplex assays—for detection of several genes in the same reaction—based on specific probes with different-coloured labels, provided that all targeted genes are amplified with similar efficiency. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers
, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction.
The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher
of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease
activity of the Taq polymerase
breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation
with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.
or PCR products on a gel or Southern blot
does not allow precise quantification. For example, over the 20-40 cycles of a typical PCR, the amount of DNA product reaches a plateau
that is not directly correlated with the amount of target DNA in the initial PCR.
Real-time PCR can be used to quantify nucleic acid
s by two methods: relative quantification and absolute quantification. Relative quantification is based on internal reference genes to determine fold-differences in expression of the target gene. Absolute quantification gives the exact number of target DNA molecules by comparison with DNA standards.
The general principle of DNA quantification by real-time PCR relies on plotting fluorescence against the number of cycles on a logarithmic scale
. A threshold for detection of DNA-based fluorescence is set slightly above background. The number of cycles at which the fluorescence exceeds the threshold is called the cycle threshold, Ct. During the exponential amplification phase, the sequence of the DNA target doubles every cycle. For example, a DNA sample whose Ct precedes that of another sample by 3 cycles contained 23 = 8 times more template. However, the efficiency of amplification is often variable among primers and templates. Therefore, the efficiency of a primer-template combination is assessed in a titration
experiment with serial dilutions of DNA template to create a standard curve
of the change in Ct with each dilution. The slope
of the linear regression
is then used to determine the efficiency of amplification, which is 100% if a dilution of 1:2 results in a Ct difference of 1.
To quantify gene expression, the Ct for an RNA or DNA from the gene of interest is divided by Ct of RNA/DNA from a housekeeping gene in the same sample to normalize for variation in the amount and quality of RNA between different samples. This normalization procedure is commonly called the ΔΔCt-method and permits comparison of expression of a gene of interest among different samples. However, for such comparison, expression of the normalizing reference gene needs to be very similar across all the samples. Choosing a reference gene fulfilling this criterion is therefore of high importance, and often challenging, because only very few genes show equal levels of expression across a range of different conditions or tissues.
Mechanism-based qPCR quantification methods have also been suggested, and have the advantage that they do not require a standard curve for quantification. Methods such as MAK2 have been shown to have equal or better quantitative performance to standard curve methods. These mechanism-based methods use knowledge about the polymerase amplification process to generate estimates of the original sample concentration.
. It is commonly used for both diagnostic and basic research
.
Diagnostic real-time PCR is applied to rapidly detect nucleic acid
s that are diagnostic of, for example, infectious diseases, cancer
and genetic abnormalities. The introduction of real-time PCR assays to the clinical microbiology laboratory has significantly improved the diagnosis of infectious diseases, and is deployed as a tool to detect newly emerging diseases, such as new strains of flu
, in diagnostic test
s.
In research settings, real-time PCR is mainly used to provide quantitative measurements of gene transcription. The technology may be used in determining how the genetic expression of a particular gene changes over time, such as in the response of tissue and cell cultures to an administration of a pharmacological
agent, progression of cell differentiation, or in response to changes in environmental conditions.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/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
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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
Intercalation (chemistry)
In chemistry, intercalation is the reversible inclusion of a molecule between two other molecules . Examples include DNA intercalation and graphite intercalation compounds.- DNA intercalation :...
with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
s that are labeled with a fluorescent
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
reporter which permits detection only after hybridization of the probe with its complementary DNA target.
Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
and Non-coding RNA
Non-coding RNA
A non-coding RNA is a functional RNA molecule that is not translated into a protein. Less-frequently used synonyms are non-protein-coding RNA , non-messenger RNA and functional RNA . The term small RNA is often used for short bacterial ncRNAs...
in cells or tissues.
Abbreviations used for real-time PCR methods vary widely and include: RTQ-PCR, Q-PCR or qPCR. Real-time reverse-transcription PCR is often denoted as: qRT-PCR, RRT-PCR, or RT-rt PCR. The acronym "RT-PCR" commonly denotes reverse-transcription PCR and not real-time PCR, but not all authors adhere to this convention.
Background
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
and turnover of gene transcripts (messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
, abbreviated to mRNA), and the number of copies of an mRNA transcript of a gene in a cell or tissue is determined by the rates of its expression and degradation.
Older methods were used to measure mRNA abundance: Differential display
Differential display
Differential display is the technique where a researcher can compare and identify changes in gene expression at the mRNA level between any pair of eukaryotic cell samples. The assay may be extended to more than one pair, if needed...
, RNAse protection assay and Northern blot
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...
. Northern blotting
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...
is often used to estimate the expression level of a gene by visualizing the abundance of its mRNA transcript in a sample. In this method, purified RNA is separated by agarose gel electrophoresis, transferred to a solid matrix (such as a nylon membrane), and probed with a specific DNA or RNA probe
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...
that is complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
to the gene of interest. Although this technique is still used to assess gene expression, it requires relatively large amounts of RNA and provides only qualitative or semiquantitative information of mRNA levels.
In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
is a common method for amplifying DNA; for mRNA-based PCR the RNA sample is first reverse transcribed to cDNA
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
with reverse transcriptase
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
.
Development of PCR technologies based on reverse transcription
Reverse transcription polymerase chain reaction
Reverse transcription polymerase chain reaction is a variant of polymerase chain reaction , a laboratory technique commonly used in molecular biology to generate many copies of a DNA sequence, a process termed "amplification"...
and fluorophore
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
s permits measurement of DNA amplification during PCR in real time, i.e., the amplified product is measured at each PCR cycle. The data thus generated can be analysed by computer software to calculate relative gene expression in several samples, or mRNA copy number. Real-time PCR can also be applied to the detection and quantification of DNA in samples to determine the presence and abundance of a particular DNA sequence in these samples.
Real-time PCR with double-stranded DNA-binding dyes as reporters
A DNA-binding dye binds to all double-stranded (ds)DNADNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNA dyes such as SYBR Green
SYBR Green
SYBR Green I is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light and emits green light . The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower...
will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer
Primer dimer
A Primer dimer is a potential by-product in PCR, a common biotechnological method. As its name implies, a PD consists of primer molecules that have attached to each other because of strings of complementary bases in the primers...
). This can potentially interfere with, or prevent, accurate quantification of the intended target sequence.
- The reaction is prepared as usual, with the addition of fluorescent dsDNA dye.
- The reaction is run in a real-time PCR machine, and after each cycle, the levels of fluorescence are measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). With reference to a standard dilution, the dsDNA concentration in the PCR can be determined.
Like other real-time PCR methods, the values obtained do not have absolute units associated with them (i.e., mRNA copies/cell). As described above, a comparison of a measured DNA/RNA sample to a standard dilution will only give a fraction or ratio of the sample relative to the standard, allowing only relative comparisons between different tissues or experimental conditions. To ensure accuracy in the quantification, it is usually necessary to normalize expression of a target gene to a stably expressed gene (see below). This can correct possible differences in RNA quantity or quality across experimental samples.
Fluorescent reporter probe method
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
reporter probes detect only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fluorescent probes can be used in multiplex assays—for detection of several genes in the same reaction—based on specific probes with different-coloured labels, provided that all targeted genes are amplified with similar efficiency. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers
Primer dimer
A Primer dimer is a potential by-product in PCR, a common biotechnological method. As its name implies, a PD consists of primer molecules that have attached to each other because of strings of complementary bases in the primers...
, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction.
The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher
Quenching (fluorescence)
Quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. As a consequence, quenching is often heavily dependent on...
of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease
Exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...
activity of the Taq polymerase
Taq polymerase
thumb|228px|right|Structure of Taq DNA polymerase bound to a DNA octamerTaq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965...
breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation
Excited state
Excitation is an elevation in energy level above an arbitrary baseline energy state. In physics there is a specific technical definition for energy level which is often associated with an atom being excited to an excited state....
with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.
- The PCR is prepared as usual (see PCR), and the reporter probe is added.
- As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.
- Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3'-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.
- Fluorescence is detected and measured in a real-time PCR machine, and its geometric increase corresponding to exponential increase of the product is used to determine the threshold cycle (CT) in each reaction.
Quantification
Quantifying gene expression by traditional DNA detection methods is unreliable. Detection of mRNA on a Northern blotNorthern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...
or PCR products on a gel or Southern blot
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
does not allow precise quantification. For example, over the 20-40 cycles of a typical PCR, the amount of DNA product reaches a plateau
Plateau (mathematics)
A plateau of a function is a part of its domain where the function has constant value.More formally, let U, V be topological spaces. A plateau for a function f: U → V is a path-connected set of points P of U such that for some y we havefor all p in P....
that is not directly correlated with the amount of target DNA in the initial PCR.
Real-time PCR can be used to quantify nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s by two methods: relative quantification and absolute quantification. Relative quantification is based on internal reference genes to determine fold-differences in expression of the target gene. Absolute quantification gives the exact number of target DNA molecules by comparison with DNA standards.
The general principle of DNA quantification by real-time PCR relies on plotting fluorescence against the number of cycles on a logarithmic scale
Logarithmic scale
A logarithmic scale is a scale of measurement using the logarithm of a physical quantity instead of the quantity itself.A simple example is a chart whose vertical axis increments are labeled 1, 10, 100, 1000, instead of 1, 2, 3, 4...
. A threshold for detection of DNA-based fluorescence is set slightly above background. The number of cycles at which the fluorescence exceeds the threshold is called the cycle threshold, Ct. During the exponential amplification phase, the sequence of the DNA target doubles every cycle. For example, a DNA sample whose Ct precedes that of another sample by 3 cycles contained 23 = 8 times more template. However, the efficiency of amplification is often variable among primers and templates. Therefore, the efficiency of a primer-template combination is assessed in a titration
Titration
Titration, also known as titrimetry, is a common laboratory method of quantitative chemical analysis that is used to determine the unknown concentration of an identified analyte. Because volume measurements play a key role in titration, it is also known as volumetric analysis. A reagent, called the...
experiment with serial dilutions of DNA template to create a standard curve
Standard curve
A standard curve is a quantitative research tool, a method of plotting assay data that is used to determine the concentration of a substance, particularly proteins and DNA. It can be used in many biological experiments...
of the change in Ct with each dilution. The slope
Slope
In mathematics, the slope or gradient of a line describes its steepness, incline, or grade. A higher slope value indicates a steeper incline....
of the linear regression
Linear regression
In statistics, linear regression is an approach to modeling the relationship between a scalar variable y and one or more explanatory variables denoted X. The case of one explanatory variable is called simple regression...
is then used to determine the efficiency of amplification, which is 100% if a dilution of 1:2 results in a Ct difference of 1.
To quantify gene expression, the Ct for an RNA or DNA from the gene of interest is divided by Ct of RNA/DNA from a housekeeping gene in the same sample to normalize for variation in the amount and quality of RNA between different samples. This normalization procedure is commonly called the ΔΔCt-method and permits comparison of expression of a gene of interest among different samples. However, for such comparison, expression of the normalizing reference gene needs to be very similar across all the samples. Choosing a reference gene fulfilling this criterion is therefore of high importance, and often challenging, because only very few genes show equal levels of expression across a range of different conditions or tissues.
Mechanism-based qPCR quantification methods have also been suggested, and have the advantage that they do not require a standard curve for quantification. Methods such as MAK2 have been shown to have equal or better quantitative performance to standard curve methods. These mechanism-based methods use knowledge about the polymerase amplification process to generate estimates of the original sample concentration.
Applications of real-time polymerase chain reaction
There are numerous applications for real-time polymerase chain reaction in the laboratoryLaboratory
A laboratory is a facility that provides controlled conditions in which scientific research, experiments, and measurement may be performed. The title of laboratory is also used for certain other facilities where the processes or equipment used are similar to those in scientific laboratories...
. It is commonly used for both diagnostic and basic research
Basic Research
Basic Research is an herbal supplement and cosmetics manufacturer based in Salt Lake City, Utah that distributes products through a large number of subsidiaries. In addition, their products are sold domestically and internationally through a number of high-end retailers. Dennis Gay is the...
.
Diagnostic real-time PCR is applied to rapidly detect nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s that are diagnostic of, for example, infectious diseases, cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
and genetic abnormalities. The introduction of real-time PCR assays to the clinical microbiology laboratory has significantly improved the diagnosis of infectious diseases, and is deployed as a tool to detect newly emerging diseases, such as new strains of flu
Influenza
Influenza, commonly referred to as the flu, is an infectious disease caused by RNA viruses of the family Orthomyxoviridae , that affects birds and mammals...
, in diagnostic test
In vitro diagnostics
In vitro diagnostic tests are medical devices intended to perform diagnoses from assays in a test tube, or more generally in a controlled environment outside a living organism...
s.
In research settings, real-time PCR is mainly used to provide quantitative measurements of gene transcription. The technology may be used in determining how the genetic expression of a particular gene changes over time, such as in the response of tissue and cell cultures to an administration of a pharmacological
Pharmacology
Pharmacology is the branch of medicine and biology concerned with the study of drug action. More specifically, it is the study of the interactions that occur between a living organism and chemicals that affect normal or abnormal biochemical function...
agent, progression of cell differentiation, or in response to changes in environmental conditions.
Further reading
- Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R. (1992). "Simultaneous amplification and detection of specific DNA sequences." Biotechnology 10:413–417.
- Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993). "Kinetic PCR: Real time monitoring of DNA amplification reactions." Biotechnology 11:1026–1030.
- Mackay, I. M. (2007). "Real-Time PCR in Microbiology." Caister Academic Press. ISBN 978-1-904455-18-9
- Wawrik B, Paul JH, Tabita FR (2002) Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes. Appl. Environ. Microbiol. 68:3771-3779.
External links
- Real Time PCR Tutorial by Dr Margaret Hunt, University of South Carolina, September 5, 2006
- openwetware
- RefGenes Open Access online tool to identify tissue specific reference genes for RT-qPCR