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A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of
molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...
based on the
polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
(PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR. Real-time PCR can be used quantitatively (quantitative real-time PCR) and semi-quantitatively (i.e., above/below a certain amount of DNA molecules) (semi-quantitative real-time PCR). Two common methods for the detection of PCR products in real-time PCR are (1) non-specific
fluorescent dye A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with sev ...
s that
intercalate Intercalation may refer to: *Intercalation (chemistry), insertion of a molecule (or ion) into layered solids such as graphite *Intercalation (timekeeping), insertion of a leap day, week or month into some calendar years to make the calendar follo ...
with any double-stranded DNA and (2) sequence-specific DNA probes consisting of
oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...
s that are labelled with a
fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
reporter, which permits detection only after hybridization of the probe with its complementary sequence. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments ( MIQE) guidelines propose that the abbreviation ''qPCR'' be used for quantitative real-time PCR and that ''RT-qPCR'' be used for reverse transcription–qPCR. The acronym "RT-PCR" commonly denotes
reverse transcription polymerase chain reaction Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
and not real-time PCR, but not all authors adhere to this convention.


Background

Cells in all organisms regulate
gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. T ...
by turnover of gene transcripts (single stranded
RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
): The amount of an expressed gene in a cell can be measured by the number of copies of an RNA transcript of that gene present in a sample. 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 The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
(PCR) is a common method for amplifying DNA; for RNA-based PCR the RNA sample is first reverse-transcribed to
complementary DNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
(cDNA) with reverse transcriptase. In order to amplify small amounts of DNA, the same methodology is used as in conventional PCR using a DNA template, at least one pair of specific primers,
deoxyribonucleotide A deoxyribonucleotide is a nucleotide that contains deoxyribose. They are the monomeric units of the informational biopolymer, deoxyribonucleic acid ( DNA). Each deoxyribonucleotide comprises three parts: a deoxyribose sugar (monosaccharide), a ni ...
triphosphates, a suitable
buffer solution A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is ...
and a thermo-stable
DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
. A substance marked with a
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with s ...
is added to this mixture in a
thermal cycler The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). Thermal cyclers may also be used in laboratories to fa ...
that contains
sensor A sensor is a device that produces an output signal for the purpose of sensing a physical phenomenon. In the broadest definition, a sensor is a device, module, machine, or subsystem that detects events or changes in its environment and sends ...
s for measuring the
fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
of the fluorophore after it has been excited at the required
wavelength In physics, the wavelength is the spatial period of a periodic wave—the distance over which the wave's shape repeats. It is the distance between consecutive corresponding points of the same phase on the wave, such as two adjacent crests, tr ...
allowing the generation rate to be measured for one or more specific products. This allows the rate of generation of the amplified product to be measured at each PCR cycle. The data thus generated can be analysed by computer software to calculate ''relative gene expression'' (or ''mRNA copy number'') in several samples. Quantitative 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. This measurement is made after each amplification cycle, and this is the reason why this method is called real time PCR (that is, immediate or simultaneous PCR). Quantitative PCR and
DNA microarray A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to ...
are modern methodologies for studying
gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. T ...
. Older methods were used to measure mRNA abundance: differential display, RNase protection assay and
northern blot The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.Ke ...
. 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 A complement is something that completes something else. Complement may refer specifically to: The arts * Complement (music), an interval that, when added to another, spans an octave ** Aggregate complementation, the separation of pitch-class ...
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 semi quantitative information of mRNA levels. Estimation errors arising from variations in the quantification method can be the result of DNA integrity, enzyme efficiency and many other factors. For this reason a number of
standardization Standardization or standardisation is the process of implementing and developing technical standards based on the consensus of different parties that include firms, users, interest groups, standards organizations and governments. Standardization ...
systems (often called normalization methods) have been developed. Some have been developed for quantifying total gene expression, but the most common are aimed at quantifying the specific gene being studied in relation to another gene called a normalizing gene, which is selected for its almost constant level of expression. These genes are often selected from housekeeping genes as their functions related to basic cellular survival normally imply constitutive gene expression. This enables researchers to report a ratio for the expression of the genes of interest divided by the expression of the selected normalizer, thereby allowing comparison of the former without actually knowing its absolute level of expression. The most commonly used normalizing genes are those that code for the following molecules:
tubulin Tubulin in molecular biology can refer either to the tubulin protein superfamily of globular proteins, or one of the member proteins of that superfamily. α- and β-tubulins polymerize into microtubules, a major component of the eukaryotic cytoske ...
, glyceraldehyde-3-phosphate dehydrogenase,
albumin Albumin is a family of globular proteins, the most common of which are the serum albumins. All the proteins of the albumin family are water- soluble, moderately soluble in concentrated salt solutions, and experience heat denaturation. Albumin ...
,
cyclophilin Cyclophilins (CYPs) are a family of proteins named after their ability to bind to ciclosporin (cyclosporin A), an immunosuppressant which is usually used to suppress rejection after internal organ transplants. They are found in all domains of lif ...
, and
ribosomal RNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal ...
s.


Basic principles

Real-time PCR is carried out in a
thermal cycler The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). Thermal cyclers may also be used in laboratories to fa ...
with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with s ...
. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the
nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
s and
DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
. The PCR process generally consists of a series of temperature changes that are repeated 25–50 times. These cycles normally consist of three stages: the first, at around 95 °C, allows the separation of the nucleic acid's double chain; the second, at a temperature of around 50–60 °C, allows the binding of the primers with the DNA template; the third, at between 68 and 72 °C, facilitates the
polymerization In polymer chemistry, polymerization (American English), or polymerisation (British English), is a process of reacting monomer molecules together in a chemical reaction to form polymer chains or three-dimensional networks. There are many for ...
carried out by the DNA polymerase. Due to the small size of the fragments the last step is usually omitted in this type of PCR as the enzyme is able to replicate the DNA
amplicon In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events. It can be formed artificially, using various methods including polymerase chain reactions (PCR) or ligase chai ...
during the change between the alignment stage and the denaturing stage. In addition, in four-step PCR the fluorescence is measured during short temperature phases lasting only a few seconds in each cycle, with a temperature of, for example, 80 °C, in order to reduce the signal caused by the presence of primer dimers when a non-specific dye is used. The temperatures and the timings used for each cycle depend on a wide variety of parameters, such as: the enzyme used to synthesize the DNA, the concentration of divalent ions and
deoxyribonucleotide A deoxyribonucleotide is a nucleotide that contains deoxyribose. They are the monomeric units of the informational biopolymer, deoxyribonucleic acid ( DNA). Each deoxyribonucleotide comprises three parts: a deoxyribose sugar (monosaccharide), a ni ...
triphosphates (dNTPs) in the reaction and the bonding temperature of the primers.


Chemical classification

Real-time PCR technique can be classified by the chemistry used to detect the PCR product, specific or non-specific fluorochromes.


Non-specific detection: real-time PCR with double-stranded DNA-binding dyes as reporters

A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, increasing the fluorescence quantum yield of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity measured at each cycle. 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 monitoring of the intended target sequence. In real-time PCR with dsDNA dyes the reaction is prepared as usual, with the addition of fluorescent dsDNA dye. Then the reaction is run in a real-time PCR instrument, and after each cycle, the intensity of fluorescence is measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). This method has the advantage of only needing a pair of primers to carry out the amplification, which keeps costs down; multiple target sequences can be monitored in a tube by using different types of dyes.


Specific detection: fluorescent reporter probe method

Fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
reporter probes detect only the DNA containing the sequence complementary to the probe; therefore, use of the reporter probe significantly increases specificity, and enables performing the technique even in the presence of other dsDNA. Using different-coloured labels, fluorescent probes can be used in multiplex assays for monitoring several target sequences in the same tube. 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 Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is t ...
activity of the
Taq polymerase ''Taq'' polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism ''Thermus aquaticus,'' from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to ''Taq'' or ''Ta ...
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. #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 quantification cycle (Cq) in each reaction.


Fusion temperature analysis

Real-time PCR permits the identification of specific, amplified DNA fragments using analysis of their melting temperature (also called ''Tm'' value, from m''elting'' t''emperature''). The method used is usually PCR with double-stranded DNA-binding dyes as reporters and the dye used is usually SYBR Green. The DNA melting temperature is specific to the amplified fragment. The results of this technique are obtained by comparing the dissociation curves of the analysed DNA samples. Unlike conventional PCR, this method avoids the previous use of
electrophoresis Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fi ...
techniques to demonstrate the results of all the samples. This is because, despite being a kinetic technique, quantitative PCR is usually evaluated at a distinct end point. The technique therefore usually provides more rapid results and/or uses fewer reactants than electrophoresis. If subsequent electrophoresis is required it is only necessary to test those samples that real time PCR has shown to be doubtful and/or to ratify the results for samples that have tested positive for a specific determinant.


Modeling

Unlike end point PCR (conventional PCR), real time PCR allows monitoring of the desired product at any point in the amplification process by measuring fluorescence (in real time frame, measurement is made of its level over a given threshold). A commonly employed method of DNA quantification by real-time PCR relies on plotting fluorescence against the number of cycles on a
logarithmic scale A logarithmic scale (or log scale) is a way of displaying numerical data over a very wide range of values in a compact way—typically the largest numbers in the data are hundreds or even thousands of times larger than the smallest numbers. Such a ...
. A threshold for detection of DNA-based fluorescence is set 3–5 times of the
standard deviation In statistics, the standard deviation is a measure of the amount of variation or dispersion of a set of values. A low standard deviation indicates that the values tend to be close to the mean (also called the expected value) of the set, whil ...
of the signal noise above background. The number of cycles at which the fluorescence exceeds the threshold is called the threshold cycle (Ct) or, according to the MIQE guidelines, quantification cycle (Cq). During the exponential amplification phase, the quantity of the target DNA template (amplicon) doubles every cycle. For example, a DNA sample whose Cq 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 (Cq) with each dilution. The
slope In mathematics, the slope or gradient of a line is a number that describes both the ''direction'' and the ''steepness'' of the line. Slope is often denoted by the letter ''m''; there is no clear answer to the question why the letter ''m'' is used ...
of the
linear regression In statistics, linear regression is a Linearity, linear approach for modelling the relationship between a Scalar (mathematics), scalar response and one or more explanatory variables (also known as dependent and independent variables). The case of ...
is then used to determine the efficiency of amplification, which is 100% if a dilution of 1:2 results in a (Cq) difference of 1. The cycle threshold method makes several assumptions of reaction mechanism and has a reliance on data from low signal-to-noise regions of the amplification profile that can introduce substantial variance during the data analysis. To quantify gene expression, the (Cq) for an RNA or DNA from the gene of interest is subtracted from the (Cq) 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 ''ΔCt-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. Although cycle threshold analysis is integrated with many commercial software systems, there are more accurate and reliable methods of analysing amplification profile data that should be considered in cases where reproducibility is a concern. 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. An extension of this approach includes an accurate model of the entire PCR reaction profile, which allows for the use of high signal-to-noise data and the ability to validate data quality prior to analysis. According to research of Ruijter et al. MAK2 assumes constant amplification efficiency during the PCR reaction. However, theoretical analysis of polymerase chain reaction, from which MAK2 was derived, has revealed that amplification efficiency is not constant throughout PCR. While MAK2 quantification provides reliable estimates of target DNA concentration in a sample under normal qPCR conditions, MAK2 does not reliably quantify target concentration for qPCR assays with competimeters.


Applications

There are numerous applications for quantitative polymerase chain reaction in the
laboratory A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratory services are provided in a variety of settings: physicia ...
. It is commonly used for both
diagnostic Diagnosis is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in many different disciplines, with variations in the use of logic, analytics, and experience, to determine "cause and effect". In systems engineer ...
and
basic research Basic research, also called pure research or fundamental research, is a type of scientific research with the aim of improving scientific theories for better understanding and prediction of natural or other phenomena. In contrast, applied resea ...
. Uses of the technique in industry include the quantification of microbial load in foods or on vegetable matter, the detection of GMOs (
genetically modified organisms A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with ...
) and the quantification and genotyping of human viral pathogens.


Quantification of gene expression

Quantifying gene expression by traditional DNA detection methods is unreliable. Detection of
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during t ...
on a
northern blot The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.Ke ...
or PCR products on a
gel A gel is a semi-solid that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state, although the liquid phase may still di ...
or
Southern blot A Southern blot is a method 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 detecti ...
does not allow precise quantification. For example, over the 20–40 cycles of a typical PCR, the amount of DNA product reaches a
plateau In geology and physical geography, a plateau (; ; ), also called a high plain or a tableland, is an area of a highland consisting of flat terrain that is raised sharply above the surrounding area on at least one side. Often one or more sides h ...
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 acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
s by two common methods: relative quantification and absolute quantification. Absolute quantification gives the exact number of target DNA molecules by comparison with DNA standards using a calibration curve. It is therefore essential that the PCR of the sample and the standard have the same amplification efficiency. Relative quantification is based on internal reference genes to determine fold-differences in expression of the target gene. The quantification is expressed as the change in expression levels of mRNA interpreted as
complementary DNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
(cDNA, generated by
reverse transcription A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
of mRNA). Relative quantification is easier to carry out as it does not require a calibration curve as the amount of the studied gene is compared to the amount of a control reference gene. As the units used to express the results of relative quantification are unimportant the results can be compared across a number of different RTqPCR. The reason for using one or more housekeeping genes is to correct non-specific variation, such as the differences in the quantity and quality of RNA used, which can affect the efficiency of reverse transcription and therefore that of the whole PCR process. However, the most crucial aspect of the process is that the reference gene must be stable. The selection of these reference genes was traditionally carried out in
molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...
using qualitative or semi-quantitative studies such as the visual examination of RNA gels, northern blot
densitometry Densitometry is the quantitative measurement of optical density in light-sensitive materials, such as photographic paper or photographic film, due to exposure to light. Overview Optical density is a result of the darkness of a developed picture ...
or semi-quantitative PCR (PCR mimics). Now, in the
genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...
era, it is possible to carry out a more detailed estimate for many organisms using transcriptomic technologies. However, research has shown that amplification of the majority of reference genes used in quantifying the expression of mRNA varies according to experimental conditions. It is therefore necessary to carry out an initial statistically sound methodological study in order to select the most suitable reference gene. A number of statistical algorithms have been developed that can detect which gene or genes are most suitable for use under given conditions. Those like geNORM or BestKeeper can compare pairs or
geometric mean In mathematics, the geometric mean is a mean or average which indicates a central tendency of a set of numbers by using the product of their values (as opposed to the arithmetic mean which uses their sum). The geometric mean is defined as the ...
s for a matrix of different reference genes and tissues.


Diagnostic uses

Diagnostic qualitative PCR is applied to rapidly detect
nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
s that are diagnostic of, for example,
infectious diseases An infection is the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmissible disease or communicable dise ...
,
cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal bl ...
and genetic abnormalities. The introduction of qualitative 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, commonly known as "the flu", is an infectious disease caused by influenza viruses. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptom ...
and
coronavirus Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans and birds, they cause respiratory tract infections that can range from mild to lethal. Mild illnesses in humans include some cases of the c ...
, in
diagnostic test A medical test is a medical procedure performed to detect, diagnose, or monitor diseases, disease processes, susceptibility, or to determine a course of treatment. Medical tests such as, physical and visual exams, diagnostic imaging, genetic ...
s.


Microbiological uses

Quantitative PCR is also used by microbiologists working in the fields of food safety, food spoilage and fermentation and for the microbial risk assessment of water quality (drinking and recreational waters) and in public health protection. qPCR may also be used to amplify taxonomic or functional markers of genes in DNA taken from environmental samples. Markers are represented by genetic fragments of DNA or complementary DNA. By amplifying a certain genetic element, one can quantify the amount of the element in the sample prior to amplification. Using taxonomic markers (ribosomal genes) and qPCR can help determine the amount of microorganisms in a sample, and can identify different families, genera, or species based on the specificity of the marker. Using functional markers (protein-coding genes) can show gene expression within a community, which may reveal information about the environment.


Detection of phytopathogens

The agricultural industry is constantly striving to produce plant propagules or seedlings that are free of pathogens in order to prevent economic losses and safeguard health. Systems have been developed that allow detection of small amounts of the DNA of ''
Phytophthora ramorum ''Phytophthora ramorum'' is the oomycete (a type of protist) plant pathogen known to cause the disease sudden oak death (SOD). The disease kills oak and other species of trees and has had devastating effects on the oak populations in California a ...
'', an oomycete that kills
oak An oak is a tree or shrub in the genus ''Quercus'' (; Latin "oak tree") of the beech family, Fagaceae. There are approximately 500 extant species of oaks. The common name "oak" also appears in the names of species in related genera, notably ...
s and other species, mixed in with the DNA of the host plant. Discrimination between the DNA of the pathogen and the plant is based on the amplification of ITS sequences, spacers located in
ribosomal RNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal ...
gene's coding area, which are characteristic for each taxon. Field-based versions of this technique have also been developed for identifying the same pathogen.


Detection of genetically modified organisms

qPCR using reverse transcription (RT-qPCR) can be used to detect GMOs given its sensitivity and dynamic range in detecting DNA. Alternatives such as DNA or protein analysis are usually less sensitive. Specific primers are used that amplify not the transgene but the promoter, terminator or even intermediate sequences used during the process of engineering the vector. As the process of creating a transgenic plant normally leads to the insertion of more than one copy of the transgene its quantity is also commonly assessed. This is often carried out by relative quantification using a control gene from the treated species that is only present as a single copy.


Clinical quantification and genotyping

Viruses can be present in humans due to direct infection or co-infections which makes
diagnosis Diagnosis is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in many different disciplines, with variations in the use of logic, analytics, and experience, to determine "cause and effect". In systems enginee ...
difficult using classical techniques and can result in an incorrect
prognosis Prognosis ( Greek: πρόγνωσις "fore-knowing, foreseeing") is a medical term for predicting the likely or expected development of a disease, including whether the signs and symptoms will improve or worsen (and how quickly) or remain stabl ...
and treatment. The use of qPCR allows both the quantification and genotyping (characterization of the strain, carried out using melting curves) of a virus such as the
hepatitis B virus ''Hepatitis B virus'' (HBV) is a partially double-stranded DNA virus, a species of the genus ''Orthohepadnavirus'' and a member of the ''Hepadnaviridae'' family of viruses. This virus causes the disease hepatitis B. Disease Despite there bein ...
. The degree of infection, quantified as the copies of the viral genome per unit of the patient's tissue, is relevant in many cases; for example, the probability that the type 1
herpes simplex virus Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), also known by their taxonomical names '' Human alphaherpesvirus 1'' and '' Human alphaherpesvirus 2'', are two members of the human ''Herpesviridae'' family, a set of viruses that produce viral in ...
reactivates is related to the number of infected
neuron A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozo ...
s in the
ganglia A ganglion is a group of neuron cell bodies in the peripheral nervous system. In the somatic nervous system this includes dorsal root ganglia and trigeminal ganglia among a few others. In the autonomic nervous system there are both sympathe ...
. This quantification is carried out either with reverse transcription or without it, as occurs if the virus becomes integrated in the human genome at any point in its cycle, such as happens in the case of HPV (human papillomavirus), where some of its variants are associated with the appearance of
cervical cancer Cervical cancer is a cancer arising from the cervix. It is due to the abnormal growth of cells that have the ability to invade or spread to other parts of the body. Early on, typically no symptoms are seen. Later symptoms may include abnormal va ...
. Real-time PCR has also brought the quantization of
human cytomegalovirus ''Human betaherpesvirus 5'', also called human cytomegalovirus (HCMV), is species of virus in the genus ''Cytomegalovirus'', which in turn is a member of the viral family known as ''Herpesviridae'' or herpesviruses. It is also commonly called ...
(CMV) which is seen in patients who are immunosuppressed following solid organ or bone marrow transplantation.


References


Bibliography

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