Reverse transcription polymerase chain reaction

Last updated
RT-PCR RT PCR Model.jpg
RT-PCR

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 chain reaction (PCR). [1] It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Confusion can arise because some authors use the acronym RT-PCR to denote real-time PCR. In this article, RT-PCR will denote Reverse Transcription PCR. Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.

Contents

The close association between RT-PCR and qPCR has led to metonymic use of the term qPCR to mean RT-PCR. Such use may be confusing, [2] as RT-PCR can be used without qPCR, for example to enable molecular cloning, sequencing or simple detection of RNA. Conversely, qPCR may be used without RT-PCR, for example, to quantify the copy number of a specific piece of DNA.

Nomenclature

The combined RT-PCR and qPCR technique has been described as quantitative RT-PCR [3] or real-time RT-PCR [4] (sometimes even called quantitative real-time RT-PCR [5] ), has been variously abbreviated as qRT-PCR, [6] RT-qPCR, [7] RRT-PCR, [8] and rRT-PCR. [9] In order to avoid confusion, the following abbreviations will be used consistently throughout this article:

TechniqueAbbreviation
Polymerase chain reactionPCR
Reverse transcription polymerase chain reactionRT-PCR
Real-time polymerase chain reactionqPCR
RT-PCR / qPCR combined techniqueqRT-PCR

Not all authors, especially earlier ones, use this convention and the reader should be cautious when following links. RT-PCR has been used to indicate both real-time PCR (qPCR) and reverse transcription PCR (RT-PCR).

History

Since its introduction in 1977, Northern blot has been used extensively for RNA quantification despite its shortcomings: (a) time-consuming technique, (b) requires a large quantity of RNA for detection, and (c) quantitatively inaccurate in the low abundance of RNA content. [10] [11] However, since PCR was invented by Kary Mullis in 1983, RT PCR has since displaced Northern blot as the method of choice for RNA detection and quantification. [12]

RT-PCR has risen to become the benchmark technology for the detection and/or comparison of RNA levels for several reasons: (a) it does not require post PCR processing, (b) a wide range (>107-fold) of RNA abundance can be measured, and (c) it provides insight into both qualitative and quantitative data. [5] Due to its simplicity, specificity and sensitivity, RT-PCR is used in a wide range of applications from experiments as simple as quantification of yeast cells in wine to more complex uses as diagnostic tools for detecting infectious agents such as the avian flu virus and SARS-CoV-2. [13] [14] [15]

Principles

In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase (RT). The cDNA is then used as a template for exponential amplification using PCR. The use of RT-PCR for the detection of RNA transcript has revolutionized the study of gene expression in the following important ways:

One-step RT-PCR vs two-step RT-PCR

One-step vs two-step RT-PCR One-step vs two-step RT-PCR.jpg
One-step vs two-step RT-PCR

The quantification of mRNA using RT-PCR can be achieved as either a one-step or a two-step reaction. The difference between the two approaches lies in the number of tubes used when performing the procedure. The two-step reaction requires that the reverse transcriptase reaction and PCR amplification be performed in separate tubes. The disadvantage of the two-step approach is susceptibility to contamination due to more frequent sample handling. [19] On the other hand, the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube in the one-step approach. The one-step approach is thought to minimize experimental variation by containing all of the enzymatic reactions in a single environment. It eliminates the steps of pipetting cDNA product, which is labor-intensive and prone to contamination, to PCR reaction. The further use of inhibitor-tolerant thermostable DNA polymerases, polymerase enhancers with an optimized one-step RT-PCR condition, supports the reverse transcription of the RNA from unpurified or crude samples, such as whole blood and serum. [20] [21] However, the starting RNA templates are prone to degradation in the one-step approach, and the use of this approach is not recommended when repeated assays from the same sample is required. Additionally, the one-step approach is reported to be less accurate compared to the two-step approach. It is also the preferred method of analysis when using DNA binding dyes such as SYBR Green since the elimination of primer-dimers can be achieved through a simple change in the melting temperature. Nevertheless, the one-step approach is a relatively convenient solution for the rapid detection of target RNA directly in biosensing.[ citation needed ]

End-point RT-PCR vs real-time RT-PCR

Quantification of RT-PCR products can largely be divided into two categories: end-point and real-time. [22] The use of end-point RT-PCR is preferred for measuring gene expression changes in small number of samples, but the real-time RT-PCR has become the gold standard method for validating quantitative results obtained from array analyses or gene expression changes on a global scale. [23]

End-point RT-PCR

The measurement approaches of end-point RT-PCR requires the detection of gene expression levels by the use of fluorescent dyes like ethidium bromide, [24] [25] P32 labeling of PCR products using phosphorimager, [26] or by scintillation counting. [18] End-point RT-PCR is commonly achieved using three different methods: relative, competitive and comparative. [27] [28]

Relative RT-PCR
Relative quantifications of RT-PCR involves the co-amplification of an internal control simultaneously with the gene of interest. The internal control is used to normalize the samples. Once normalized, a direct comparison of relative transcript abundances across multiple samples of mRNA can be made. One precaution to note is that the internal control must be chosen so that it is not affected by the experimental treatment. The expression level should be constant across all samples and with the mRNA of interest for the results to be accurate and meaningful. Because the quantification of the results are analyzed by comparing the linear range of the target and control amplification, it is crucial to take into consideration the starting target molecules concentration and their amplification rate prior to starting the analysis. The results of the analysis are expressed as the ratios of gene signal to internal control signal, which the values can then be used for the comparison between the samples in the estimation of relative target RNA expression. [25] [28] [29]
Competitive RT-PCR
Competitive RT-PCR technique is used for absolute quantification. It involves the use of a synthetic “competitor” RNA that can be distinguished from the target RNA by a small difference in size or sequence. It is important for the design of the synthetic RNA be identical in sequence but slightly shorter than the target RNA for accurate results. Once designed and synthesized, a known amount of the competitor RNA is added to experimental samples and is co-amplified with the target using RT-PCR. Then, a concentration curve of the competitor RNA is produced and it is used to compare the RT-PCR signals produced from the endogenous transcripts to determine the amount of target present in the sample. [28] [30]
Comparative RT-PCR
Comparative RT-PCR is similar to the competitive RT-PCR in that the target RNA competes for amplification reagents within a single reaction with an internal standard of unrelated sequence. Once the reaction is complete, the results are compared to an external standard curve to determine the target RNA concentration. In comparison to the relative and competitive quantification methods, comparative RT-PCR is considered to be the more convenient method to use since it does not require the investigator to perform a pilot experiment; in relative RT-PCR, the exponential amplification range of the mRNA must be predetermined and in competitive RT-PCR, a synthetic competitor RNA must be synthesized. [28] [31] [32] [33] [34]

Real-time RT-PCR

The emergence of novel fluorescent DNA labeling techniques in the past few years has enabled the analysis and detection of PCR products in real-time and has consequently led to the widespread adoption of real-time RT-PCR for the analysis of gene expression. [35] Not only is real-time RT-PCR now the method of choice for quantification of gene expression, it is also the preferred method of obtaining results from array analyses and gene expressions on a global scale. [36] Currently, there are four different fluorescent DNA probes available for the real-time RT-PCR detection of PCR products: SYBR Green, TaqMan, molecular beacons, and scorpion probes. All of these probes allow the detection of PCR products by generating a fluorescent signal. While the SYBR Green dye emits its fluorescent signal simply by binding to the double-stranded DNA in solution, the TaqMan probes', molecular beacons' and scorpions' generation of fluorescence depend on Förster Resonance Energy Transfer (FRET) coupling of the dye molecule and a quencher moiety to the oligonucleotide substrates. [37]

SYBR Green
When the SYBR Green binds to the double-stranded DNA of the PCR products, it will emit light upon excitation. The intensity of the fluorescence increases as the PCR products accumulate. This technique is easy to use since designing of probes is not necessary given lack of specificity of its binding. However, since the dye does not discriminate the double-stranded DNA from the PCR products and those from the primer-dimers, overestimation of the target concentration is a common problem. Where accurate quantification is an absolute necessity, further assay for the validation of results must be performed. Nevertheless, among the real-time RT-PCR product detection methods, SYBR Green is the most economical and easiest to use. [22] [23]
Taqman probes Taqman.png
Taqman probes
TaqMan probes
TaqMan probes are oligonucleotides that have a fluorescent probe attached to the 5' end and a quencher to the 3' end. During PCR amplification, these probes will hybridize to the target sequences located in the amplicon and as polymerase replicates the template with TaqMan bound, it also cleaves the fluorescent probe due to polymerase 5'- nuclease activity. Because the close proximity between the quench molecule and the fluorescent probe normally prevents fluorescence from being detected through FRET, the decoupling results in the increase of intensity of fluorescence proportional to the number of the probe cleavage cycles. Although well-designed TaqMan probes produce accurate real-time RT-PCR results, it is expensive and time-consuming to synthesize when separate probes must be made for each mRNA target analyzed. [22] [16] [38] Additionally, these probes are light sensitive and must be carefully frozen as aliquots to prevent degradation.
Molecular beacon probes
Similar to the TaqMan probes, molecular beacons also make use of FRET detection with fluorescent probes attached to the 5' end and a quencher attached to the 3' end of an oligonucleotide substrate. However, whereas the TaqMan fluorescent probes are cleaved during amplification, molecular beacon probes remain intact and rebind to a new target during each reaction cycle. When free in solution, the close proximity of the fluorescent probe and the quencher molecule prevents fluorescence through FRET. However, when molecular beacon probes hybridize to a target, the fluorescent dye and the quencher are separated resulting in the emittance of light upon excitation. As is with the TaqMan probes, molecular beacons are expensive to synthesize and require separate probes for each RNA target. [19]
Scorpion probes
The scorpion probes, like molecular beacons, will not be fluorescent active in an unhybridized state, again, due to the fluorescent probe on the 5' end being quenched by the moiety on the 3' end of an oligonucleotide. With Scorpions, however, the 3' end also contains sequence that is complementary to the extension product of the primer on the 5' end. When the Scorpion extension binds to its complement on the amplicon, the Scorpion structure opens, prevents FRET, and enables the fluorescent signal to be measured. [39]
Multiplex probes
TaqMan probes, molecular beacons, and scorpions allow the concurrent measurement of PCR products in a single tube. This is possible because each of the different fluorescent dyes can be associated with a specific emission spectra. Not only does the use of multiplex probes save time and effort without compromising test utility, its application in wide areas of research such as gene deletion analysis, mutation and polymorphism analysis, quantitative analysis, and RNA detection, make it an invaluable technique for laboratories of many discipline. [39] [40] [41]

Two strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method. [42]

Application

The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique in which a very low copy number of RNA molecules can be detected. RT-PCR is widely used in the diagnosis of genetic diseases and, semiquantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression.

Research methods

RT-PCR is commonly used in research methods to measure gene expression. For example, Lin et al. used qRT-PCR to measure expression of Gal genes in yeast cells. First, Lin et al. engineered a mutation of a protein suspected to participate in the regulation of Gal genes. This mutation was hypothesized to selectively abolish Gal expression. To confirm this, gene expression levels of yeast cells containing this mutation were analyzed using qRT-PCR. The researchers were able to conclusively determine that the mutation of this regulatory protein reduced Gal expression. [43] Northern blot analysis is used to study the RNA's gene expression further.

Gene insertion

RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. Because most eukaryotic genes contain introns, which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact (without regard to the error-prone nature of reverse transcriptases) DNA sequence that would be directly translated into protein after transcription. When these genes are expressed in prokaryotic cells for the sake of protein production or purification, the RNA produced directly from transcription need not undergo splicing as the transcript contains only exons. (Prokaryotes, such as E. coli, lack the mRNA splicing mechanism of eukaryotes).

Genetic disease diagnosis

RT-PCR can be used to diagnose genetic disease such as Lesch–Nyhan syndrome. This genetic disease is caused by a malfunction in the HPRT1 gene, which clinically leads to the fatal uric acid urinary stone and symptoms similar to gout.[6][ clarification needed ] Analyzing a pregnant mother and a fetus for mRNA expression levels of HPRT1 will reveal if the mother is a carrier and if the fetus will likely to develop Lesch–Nyhan syndrome. [44]

Cancer detection

Scientists are working on ways to use RT-PCR in cancer detection to help improve prognosis, and monitor response to therapy. Circulating tumor cells produce unique mRNA transcripts depending on the type of cancer. The goal is to determine which mRNA transcripts serve as the best biomarkers for a particular cancer cell type and then analyze its expression levels with RT-PCR. [45]

RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenzavirus A, retroviruses like HIV and SARS-CoV-2. [46]

Challenges

Despite its major advantages, RT-PCR is not without drawbacks. The exponential growth of the reverse transcribed complementary DNA (cDNA) during the multiple cycles of PCR produces inaccurate end point quantification due to the difficulty in maintaining linearity. [47] In order to provide accurate detection and quantification of RNA content in a sample, qRT-PCR was developed using fluorescence-based modification to monitor the amplification products during each cycle of PCR. The extreme sensitivity of the technique can be a double-edged sword since even the slightest DNA contamination can lead to undesirable results. [48] A simple method for elimination of false positive results is to include anchors, or tags, to the 5' region of a gene specific primer. [49] Additionally, planning and design of quantification studies can be technically challenging due to the existence of numerous sources of variation including template concentration and amplification efficiency. [31] Spiking in a known quantity of RNA into a sample, adding a series of RNA dilutions generating a standard curve, and adding in a no template copy sample (no cDNA) may used as controls.

Protocol

RT-PCR can be carried out by the one-step RT-PCR protocol or the two-step RT-PCR protocol.

One-step RT-PCR

One-step RT-PCR subjects mRNA targets (up to 6 kb) to reverse transcription followed by PCR amplification in a single test tube. Using intact, high-quality RNA and a sequence-specific primer will produce the best results.

Once a one-step RT-PCR kit with a mix of reverse transcriptase, Taq DNA polymerase, and a proofreading polymerase is selected and all necessary materials and equipment are obtained a reaction mix is to be prepared. The reaction mix includes dNTPs, primers, template RNA, necessary enzymes, and a buffer solution. The reaction mix is added to a PCR tube for each reaction, followed by template RNA. The PCR tubes are then placed in a thermal cycler to begin cycling. In the first cycle, the synthesis of cDNA occurs. The second cycle is the initial denaturation wherein reverse transcriptase is inactivated. The remaining 40-50 cycles are the amplification, which includes denaturation, annealing, and elongation. When amplification is complete, the RT-PCR products can be analyzed with gel electrophoresis. [50] [51]

(PCR Applications Manual and Biotools)

Two-step RT-PCR

Two-step RT-PCR, as the name implies, occurs in two steps. First the reverse transcription and then the PCR. This method is more sensitive than the one-step method. Kits are also useful for two-step RT-PCR. Just as for one-step PCR, use only intact, high-quality RNA for the best results. The primer for two-step PCR does not have to be sequence-specific.

Step one

First combine template RNA, primer, dNTP mix, and nuclease-free water in a PCR tube. Then, add an RNase inhibitor and reverse transcriptase to the PCR tube. Next, place the PCR tube into a thermal cycler for one cycle wherein annealing, extending, and inactivating of reverse transcriptase occurs. Finally, proceed directly to step two which is PCR, or store product on ice until PCR can be performed.

Step two

Add master mix which contains buffer, dNTP mix, MgCl2, Taq polymerase, and nuclease-free water to each PCR tube. Then add the necessary primer to the tubes. Next, place the PCR tubes in a thermal cycler for 30 cycles of the amplification program. This includes denaturation, annealing, and elongation. The products of RT-PCR can be analyzed with gel electrophoresis. [52]

Publication guidelines

Quantitative RT-PCR assay is considered to be the gold standard for measuring the number of copies of specific cDNA targets in a sample but it is poorly standardized. [53] As a result, while there are numerous publications utilizing the technique, many provide inadequate experimental detail and use unsuitable data analysis to draw inappropriate conclusions. Due to the inherent variability in the quality of any quantitative PCR data, not only do reviewers have a difficult time evaluating these manuscripts, but the studies also become impossible to replicate. [54] Recognizing the need for the standardization of the reporting of experimental conditions, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE, pronounced mykee) guidelines have been published by an international consortium of academic scientists. The MIQE guidelines describe the minimum information necessary for evaluating quantitative PCR experiments that should be required for publication to encourage better experimental practice and ensuring the relevance, accuracy, correct interpretation, and repeatability of quantitative PCR data. [55]

Besides reporting guidelines, the MIQE stresses the need to standardize the nomenclature associated with quantitative PCR to avoid confusion; for example, the abbreviation qPCR should be used for quantitative real-time PCR, while RT-qPCR should be used for reverse transcription-qPCR, and genes used for normalization should be referred to as reference genes instead of housekeeping genes . It also proposes that commercially derived terms like TaqMan probes should not be used, but instead referred to as hydrolysis probes . Additionally, it is proposed that the quantification cycle (Cq) be used to describe the PCR cycle used for quantification instead of the threshold cycle (Ct), crossing point (Cp), and takeoff point (TOP), which refer to the same value but were coined by different manufacturers of real-time instruments. [53]

The guideline consists of the following elements: 1) experimental design, 2) sample, 3) nucleic acid extraction, 4) reverse transcription, 5) qPCR target information, 6) oligonucleotides, 7) protocol, 8) validation, and 9) data analysis. Specific items within each element carry a label of either E (essential) or D (desirable). Those labeled E are considered critical and indispensable while those labeled D are considered peripheral yet important for best practices. [55]

Research

In 2023, researchers developed a working prototype of an RT-LAMP lab-on-a-chip system, which provided results for SARS-CoV-2 tests within three minutes.The technology integrates microfluidic channels into printed circuit boards with, which may enable low-cost mass production. [56] [57]

Related Research Articles

<span class="mw-page-title-main">Complementary DNA</span> DNA reverse transcribed from RNA

In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed from an RNA. cDNA exists in both single-stranded and double-stranded forms and in both natural and engineered forms.

<span class="mw-page-title-main">Polymerase chain reaction</span> Laboratory technique to multiply a DNA sample for study

The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

Viral load, also known as viral burden, is a numerical expression of the quantity of virus in a given volume of fluid, including biological and environmental specimens. It is not to be confused with viral titre or viral titer, which depends on the assay. When an assay for measuring the infective virus particle is done, viral titre often refers to the concentration of infectious viral particles, which is different from the total viral particles. Viral load is measured using body fluids sputum and blood plasma. As an example of environmental specimens, the viral load of norovirus can be determined from run-off water on garden produce. Norovirus has not only prolonged viral shedding and has the ability to survive in the environment but a minuscule infectious dose is required to produce infection in humans: less than 100 viral particles.

<span class="mw-page-title-main">Real-time polymerase chain reaction</span> Laboratory technique of molecular biology

A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.

TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann-La Roche for diagnostic assays and by Applied Biosystems for research applications.

SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.

Nucleic acid sequence-based amplification, commonly referred to as NASBA, is a method in molecular biology which is used to produce multiple copies of single stranded RNA. NASBA is a two-step process that takes RNA and anneals specially designed primers, then utilizes an enzyme cocktail to amplify it.

Digital polymerase chain reaction is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA, or RNA. The key difference between dPCR and qPCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences—such as copy number variants and point mutations.

The detection of genetically modified organisms in food or feed is possible by biochemical means. It can either be qualitative, showing which genetically modified organism (GMO) is present, or quantitative, measuring in which amount a certain GMO is present. Being able to detect a GMO is an important part of GMO labeling, as without detection methods the traceability of GMOs would rely solely on documentation.

Potato virus Y (PVY) is a plant pathogenic virus of the family Potyviridae, and one of the most important plant viruses affecting potato production.

Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of DNA for diagnostic purposes and a low-cost alternative to detect certain diseases. LAMP is an isothermal nucleic acid amplification technique. In contrast to the polymerase chain reaction (PCR) technology, in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler. LAMP was invented in 1998 by Eiken Chemical Company in Tokyo. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with a reverse transcription step to allow the detection of RNA.

Webtag is an on-line bioinformatics tool providing oligonucleotide sequences that are absent from a specified genome. These tags can be appended to gene specific primers for reverse transcriptase polymerase chain reaction (RT-PCR) experiments, circumventing genomic DNA contamination.

The versatility of polymerase chain reaction (PCR) has led to modifications of the basic protocol being used in a large number of variant techniques designed for various purposes. This article summarizes many of the most common variations currently or formerly used in molecular biology laboratories; familiarity with the fundamental premise by which PCR works and corresponding terms and concepts is necessary for understanding these variant techniques.

A primer dimer (PD) is a potential by-product in the polymerase chain reaction (PCR), a common biotechnological method. As its name implies, a PD consists of two primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification.

Multiplex polymerase chain reaction refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously. This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR.

The terms "relative fluorescence units" (RFU) and "RFU peak" refer to measurements in electrophoresis methods, such as for DNA analysis. A "relative fluorescence unit" is a unit of measurement used in analysis which employs fluorescence detection. Fluorescence is detected using a charge-coupled device (CCD) array, when the labeled fragments, which are separated within a capillary by using electrophoresis, are energized by laser light and travel across the detection window. A computer program measures the results, determining the quantity or size of the fragments, at each data point, from the level of fluorescence intensity. Samples which contain higher quantities of amplified DNA will have higher corresponding RFU values.

Stephen Andrew Bustin is a British scientist, former professor of molecular sciences at Queen Mary University of London from 2004 to 2012, as well as visiting professor at Middlesex University, beginning in 2006. In 2012 he was appointed Professor of Allied Health and Medicine at Anglia Ruskin University. He is known for his research into polymerase chain reaction, and has written a book on the topic, entitled A-Z of Quantitative PCR. This book has been called "the bible of qPCR."

Recombinase polymerase amplification (RPA) is a single tube, isothermal alternative to the polymerase chain reaction (PCR). By adding a reverse transcriptase enzyme to an RPA reaction, it can detect RNA as well as DNA, without the need for a separate step to produce cDNA. Because it is isothermal, RPA can use much simpler equipment than PCR, which requires a thermal cycler. Operating best at temperatures of 37–42 °C and still working, albeit more slowly, at room temperature means RPA reactions can in theory be run quickly by simply holding a tube in the hand. This makes RPA an excellent candidate for developing low-cost, rapid, point-of-care molecular tests. An international quality assessment of molecular detection of Rift Valley fever virus performed as well as the best RT-PCR tests, detecting less concentrated samples missed by some PCR tests and an RT-LAMP test. RPA was developed and launched by TwistDx Ltd., a biotechnology company based in Cambridge, UK.

<span class="mw-page-title-main">Reverse Transcription Loop-mediated Isothermal Amplification</span>

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one step nucleic acid amplification method to multiply specific sequences of RNA. It is used to diagnose infectious disease caused by RNA viruses.

<span class="mw-page-title-main">Thermostable DNA polymerase</span> DNA polymerases that originate from thermophiles

Thermostable DNA polymerases are DNA polymerases that originate from thermophiles, usually bacterial or archaeal species, and are therefore thermostable. They are used for the polymerase chain reaction and related methods for the amplification and modification of DNA.

References

  1. ↑ Freeman WM, Walker SJ, Vrana KE (January 1999). "Quantitative RT-PCR: pitfalls and potential". BioTechniques . 26 (1): 112–22, 124–5. doi: 10.2144/99261rv01 . PMID   9894600.
  2. ↑ Mackay, Ian (2007). Real-time PCR in Microbiology: From Diagnosis to Characterization . Norfolk, England: Caister Academic Press. pp.  440. ISBN   978-1-904455-18-9.
  3. ↑ Joyce C (2002). "Quantitative RT-PCR: A Review of Current Methodologies". RT-PCR Protocols. Methods Mol. Biol. Vol. 193. pp. 83–92. doi:10.1385/1-59259-283-X:083. ISBN   978-1-59259-283-8. PMID   12325527.
  4. ↑ Kang XP, Jiang T, Li YQ, et al. (2010). "A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus". Virol. J. 7: 113. doi: 10.1186/1743-422X-7-113 . PMC   2892456 . PMID   20515509.
  5. 1 2 Bustin SA, Benes V, Nolan T, Pfaffl MW (June 2005). "Quantitative real-time RT-PCR--a perspective". J. Mol. Endocrinol. 34 (3): 597–601. CiteSeerX   10.1.1.528.6638 . doi:10.1677/jme.1.01755. PMID   15956331. S2CID   1754364.
  6. ↑ Varkonyi-Gasic E, Hellens RP (2010). "qRT-PCR of Small RNAs". Plant Epigenetics. Methods in Molecular Biology. Vol. 631. pp. 109–22. doi:10.1007/978-1-60761-646-7_10. ISBN   978-1-60761-645-0. PMID   20204872.
  7. ↑ Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M (April 2010). "A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines". Methods. 50 (4): S1–5. doi:10.1016/j.ymeth.2010.01.005. PMID   20215014.
  8. ↑ Spackman E, Senne DA, Myers TJ, et al. (September 2002). "Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes". J. Clin. Microbiol. 40 (9): 3256–60. doi:10.1128/jcm.40.9.3256-3260.2002. PMC   130722 . PMID   12202562.
  9. ↑ "ACCELERATED EMERGENCY USE AUTHORIZATION (EUA) SUMMARY COVID-19 RT-PCR TEST (LABORATORY CORPORATION OF AMERICA)". FDA. Retrieved 3 April 2020.
  10. ↑ Alwine JC, Kemp DJ, Stark GR (December 1977). "Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5350–4. Bibcode:1977PNAS...74.5350A. doi: 10.1073/pnas.74.12.5350 . PMC   431715 . PMID   414220.
  11. ↑ Streit S, Michalski CW, Erkan M, Kleeff J, Friess H (2009). "Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues". Nat Protoc. 4 (1): 37–43. doi:10.1038/nprot.2008.216. PMID   19131955. S2CID   24980302.
  12. ↑ Bustin SA (October 2000). "Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays". J. Mol. Endocrinol. 25 (2): 169–93. doi: 10.1677/jme.0.0250169 . PMID   11013345.
  13. ↑ Hierro N, Esteve-Zarzoso B, González A, Mas A, Guillamón JM (November 2006). "Real-time quantitative PCR (QPCR) and reverse transcription-QPCR for detection and enumeration of total yeasts in wine". Appl. Environ. Microbiol. 72 (11): 7148–55. Bibcode:2006ApEnM..72.7148H. doi:10.1128/AEM.00388-06. PMC   1636171 . PMID   17088381.
  14. ↑ Slomka MJ, Pavlidis T, Coward VJ, et al. (July 2009). "Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses". Influenza and Other Respiratory Viruses. 3 (4): 151–64. doi:10.1111/j.1750-2659.2009.00083.x. PMC   4634683 . PMID   19627372.
  15. ↑ Mission summary: WHO Field Visit to Wuhan, China 20–21 January 2020: https://www.who.int/china/news/detail/22-01-2020-field-visit-wuhan-china-jan-2020
  16. 1 2 Deepak S, Kottapalli K, Rakwal R, et al. (June 2007). "Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes". Curr. Genomics. 8 (4): 234–51. doi:10.2174/138920207781386960. PMC   2430684 . PMID   18645596.
  17. 1 2 Bustin SA (August 2002). "Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems". J. Mol. Endocrinol. 29 (1): 23–39. doi: 10.1677/jme.0.0290023 . PMID   12200227.
  18. 1 2 Souazé F, Ntodou-Thomé A, Tran CY, Rostène W, Forgez P (August 1996). "Quantitative RT-PCR: limits and accuracy". BioTechniques. 21 (2): 280–5. doi: 10.2144/96212rr01 . PMID   8862813.
  19. 1 2 Wong ML, Medrano JF (July 2005). "Real-time PCR for mRNA quantitation". BioTechniques. 39 (1): 75–85. doi: 10.2144/05391rv01 . PMID   16060372.
  20. ↑ Li, Lang; He, Jian-an; Wang, Wei; Xia, Yun; Song, Li; Chen, Ze-han; Zuo, Hang-zhi; Tan, Xuan-Ping; Ho, Aaron Ho-Pui; Kong, Siu-Kai; Loo, Jacky Fong-Chuen (2019-08-01). "Development of a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for clinical Zika diagnosis". International Journal of Infectious Diseases. 85: 167–174. doi: 10.1016/j.ijid.2019.06.007 . ISSN   1201-9712. PMID   31202908.
  21. ↑ Bachofen, Claudia; Willoughby, Kim; Zadoks, Ruth; Burr, Paul; Mellor, Dominic; Russell, George C. (2013-06-01). "Direct RT-PCR from serum enables fast and cost-effective phylogenetic analysis of bovine viral diarrhoea virus". Journal of Virological Methods. 190 (1): 1–3. doi:10.1016/j.jviromet.2013.03.015. ISSN   0166-0934. PMID   23541784.
  22. 1 2 3 Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW (October 2000). "Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods". Anal. Biochem. 285 (2): 194–204. doi:10.1006/abio.2000.4753. PMID   11017702. S2CID   258810.
  23. 1 2 Rajeevan MS, Vernon SD, Taysavang N, Unger ER (February 2001). "Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR". J Mol Diagn. 3 (1): 26–31. doi:10.1016/S1525-1578(10)60646-0. PMC   1907344 . PMID   11227069.
  24. ↑ Stone-Marschat M, Carville A, Skowronek A, Laegreid WW (March 1994). "Detection of African horse sickness virus by reverse transcription-PCR". J. Clin. Microbiol. 32 (3): 697–700. doi:10.1128/JCM.32.3.697-700.1994. PMC   263109 . PMID   8195381.
  25. 1 2 Minton AP (April 1995). "Confinement as a determinant of macromolecular structure and reactivity. II. Effects of weakly attractive interactions between confined macrosolutes and confining structures". Biophys. J. 68 (4): 1311–22. Bibcode:1995BpJ....68.1311M. doi:10.1016/S0006-3495(95)80304-8. PMC   1282026 . PMID   7787020.
  26. ↑ Hsu M, Yu EY, Sprušanský O, McEachern MJ, Lue NF (July 2012). "Functional analysis of the single Est1/Ebs1 homologue in Kluyveromyces lactis reveals roles in both telomere maintenance and rapamycin resistance". Eukaryotic Cell. 11 (7): 932–42. doi:10.1128/EC.05319-11. PMC   3416500 . PMID   22544908.
  27. ↑ Schmittgen TD, Livak KJ (2008). "Analyzing real-time PCR data by the comparative C(T) method". Nat Protoc. 3 (6): 1101–8. doi:10.1038/nprot.2008.73. PMID   18546601. S2CID   205464270.
  28. 1 2 3 4 Tang, Yi-Wei (2012-09-13), Advanced Techniques in Diagnostic Microbiology, Springer, ISBN   978-1461439691
  29. ↑ Gause WC, Adamovicz J (June 1994). "The use of the PCR to quantitate gene expression". Genome Research. 3 (6): S123–35. doi: 10.1101/gr.3.6.s123 . PMID   7522722.
  30. ↑ Tsai SJ, Wiltbank MC (November 1996). "Quantification of mRNA using competitive RT-PCR with standard-curve methodology". BioTechniques. 21 (5): 862–6. doi: 10.2144/96215st04 . PMID   8922627.
  31. 1 2 Ramakers C, Ruijter JM, Deprez RH, Moorman AF (March 2003). "Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data". Neurosci. Lett. 339 (1): 62–6. doi:10.1016/S0304-3940(02)01423-4. PMID   12618301. S2CID   9459695.
  32. ↑ Halford WP, Falco VC, Gebhardt BM, Carr DJ (January 1999). "The inherent quantitative capacity of the reverse transcription-polymerase chain reaction". Anal. Biochem. 266 (2): 181–91. doi: 10.1006/abio.1998.2913 . PMID   9888974.
  33. ↑ King N (2010). "The use of comparative quantitative RT-PCR to investigate the effect of cysteine incubation on GPx1 expression in freshly isolated cardiomyocytes". RT-PCR Protocols. Methods in Molecular Biology. Vol. 630. pp. 215–32. doi:10.1007/978-1-60761-629-0_14. ISBN   978-1-60761-628-3. PMID   20301000.
  34. ↑ Chang JT, Chen IH, Liao CT, et al. (November 2002). "A reverse transcription comparative real-time PCR method for quantitative detection of angiogenic growth factors in head and neck cancer patients". Clin. Biochem. 35 (8): 591–6. doi:10.1016/S0009-9120(02)00403-4. PMID   12498992.
  35. ↑ Lu, Rou-Jian; Zhao, Li; Huang, Bao-Ying; Ye, Fei; Wang, Wen-Ling; Tan, Wen-Jie (5 September 2021). "Real-time reverse transcription-polymerase chain reaction assay panel for the detection of severe acute respiratory syndrome coronavirus 2 and its variants". Chinese Medical Journal. 134 (17): 2048–2053. doi:10.1097/CM9.0000000000001687. PMC   8439998 . PMID   34402479 . Retrieved 17 February 2023.
  36. ↑ Jawerth, Nicole (27 March 2020). "How is the COVID-19 Virus Detected using real time reverse transcription–polymerase chain reaction?". International Atomic Energy Agency . Retrieved 16 February 2023.
  37. ↑ Holden, M. J.; Wang, L. (2008). "Quantitative Real-Time PCR: Fluorescent Probe Options and Issues". Standardization and Quality Assurance in Fluorescence Measurements II. Springer Series on Fluorescence. Vol. 6. p. 489. doi:10.1007/4243_2008_046. ISBN   978-3-540-70570-3.
  38. ↑ Yang DK, Kweon CH, Kim BH, et al. (December 2004). "TaqMan reverse transcription polymerase chain reaction for the detection of Japanese encephalitis virus". J. Vet. Sci. 5 (4): 345–51. doi: 10.4142/jvs.2004.5.4.345 . PMID   15613819.
  39. 1 2 Sharkey FH, Banat IM, Marchant R (July 2004). "Detection and quantification of gene expression in environmental bacteriology". Appl. Environ. Microbiol. 70 (7): 3795–806. Bibcode:2004ApEnM..70.3795S. doi:10.1128/AEM.70.7.3795-3806.2004. PMC   444812 . PMID   15240248.
  40. ↑ Ratcliff RM, Chang G, Kok T, Sloots TP (July 2007). "Molecular diagnosis of medical viruses". Curr Issues Mol Biol. 9 (2): 87–102. PMID   17489437.
  41. ↑ Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE (October 2000). "Multiplex PCR: optimization and application in diagnostic virology". Clin. Microbiol. Rev. 13 (4): 559–70. doi:10.1128/cmr.13.4.559-570.2000. PMC   88949 . PMID   11023957.
  42. ↑ Bustin SA (July 2005). "Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences". Expert Rev. Mol. Diagn. 5 (4): 493–8. doi:10.1586/14737159.5.4.493. PMID   16013967. S2CID   1833811.
  43. ↑ Lin L, Chamberlain L, Zhu LJ, Green MR (February 2012). "Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4". Proc. Natl. Acad. Sci. U.S.A. 109 (6): 1997–2002. Bibcode:2012PNAS..109.1997L. doi: 10.1073/pnas.1116340109 . PMC   3277556 . PMID   22308403.
  44. ↑ Torres RJ, Garcia MG, Puig JG (December 2012). "Carrier and prenatal diagnosis of Lesch-Nyhan disease due to a defect in HPRT gene expression regulation". Gene. 511 (2): 306–7. doi:10.1016/j.gene.2012.09.121. PMID   23046577.
  45. ↑ Xi L, Nicastri DG, El-Hefnawy T, Hughes SJ, Luketich JD, Godfrey TE (July 2007). "Optimal markers for real-time quantitative reverse transcription PCR detection of circulating tumor cells from melanoma, breast, colon, esophageal, head and neck, and lung cancers". Clin. Chem. 53 (7): 1206–15. doi: 10.1373/clinchem.2006.081828 . PMID   17525108.
  46. ↑ "Coronavirus: il viaggio dei test". Istituto Superiore di Sanità.
  47. ↑ Shiao YH (December 2003). "A new reverse transcription-polymerase chain reaction method for accurate quantification". BMC Biotechnol. 3: 22. doi: 10.1186/1472-6750-3-22 . PMC   317330 . PMID   14664723.
  48. ↑ Gettemy JM, Ma B, Alic M, Gold MH (February 1998). "Reverse transcription-PCR analysis of the regulation of the manganese peroxidase gene family". Appl. Environ. Microbiol. 64 (2): 569–74. Bibcode:1998ApEnM..64..569G. doi:10.1128/AEM.64.2.569-574.1998. PMC   106084 . PMID   9464395.
  49. ↑ Martel, Fatima; Dirk Grundemann; Edgar Schöig (2002-03-31). "A simple method for elimination of false positive results in RT-PCR". J Biochem Mol Biol. 35 (2): 248–250. doi: 10.5483/BMBRep.2002.35.2.248 . PMID   12297038.
  50. ↑ "High Transcript Tools OneStep Kit". Biotools. Archived from the original on 20 May 2013. Retrieved 12 December 2012.
  51. ↑ Degen, Hans-Joachim; Deufel, Annette; Eisel, Doris; Grünewald-Janho, Stefanie; Keesey, Joe (2006). PCR Applications Manual (PDF) (3 ed.). Roche Diagnostics. pp. 135–137.
  52. ↑ "RT-PCR Two-Step Protocol" (PDF). MIT. Retrieved 12 December 2012.
  53. 1 2 "www.microarrays.ca" (PDF).
  54. ↑ Bustin SA (April 2010). "Why the need for qPCR publication guidelines?--The case for MIQE". Methods. 50 (4): 217–26. doi:10.1016/j.ymeth.2009.12.006. PMID   20025972.
  55. 1 2 Bustin SA, Benes V, Garson JA, et al. (April 2009). "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments". Clin. Chem. 55 (4): 611–22. doi: 10.1373/clinchem.2008.112797 . PMID   19246619.
  56. ↑ Papamatthaiou S, Boxall-Clasby J, Douglas EJ, Jajesniak P, Peyret H, Mercer-Chalmers J, Kumar VK, Lomonossoff GP, Reboud J, Laabei M, Cooper JM, Kasprzyk-Hordern B, Moschou D (October 2023). "LoCKAmp: lab-on-PCB technology for <3 minute virus genetic detection". Lab on a Chip . 23 (20): 4400–4412. doi:10.1039/d3lc00441d. PMC   10563828 . PMID   37740394.
  57. ↑ "LoCKAmp diagnosis device hailed as 'world's fastest Covid test'". The Engineer . 2 November 2023. Retrieved 29 October 2024.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.