Reverse transcription polymerase chain reaction
Encyclopedia
Reverse transcription polymerase chain reaction (RT-PCR) is a variant of polymerase chain reaction
(PCR), a laboratory technique commonly used in molecular biology
to generate many copies of a DNA
sequence, a process termed "amplification". In RT-PCR, however, an RNA strand is first reverse transcribed
into its DNA
complement (complementary DNA
, or cDNA) using the enzyme reverse transcriptase
, and the resulting cDNA is amplified using traditional PCR or real-time PCR
. Reverse transcription PCR is not to be confused with real-time polymerase chain reaction
(Q-PCR/qRT-PCR), which is also sometimes abbreviated as RT-PCR.
, which are complementary to a defined sequence on each of the two strands of the cDNA. These primers
are then extended by a DNA polymerase
and a copy of the strand is made after each cycle, leading to exponential amplification.
RT-PCR includes three major steps. The first step is reverse transcription (RT), in which RNA is reverse transcribed to cDNA using reverse transcriptase
. This step is very important in order to perform PCR since DNA polymerase can act only on DNA
templates. The RT step can be performed either in the same tube with PCR (one-step PCR) or in a separate one (two-step PCR) using a temperature between 40°C and 50°C, depending on the properties of the reverse transcriptase used.
The next step involves the denaturation of the dsDNA at 95°C, so that the two strands separate and the primers can bind again at lower temperatures and begin a new chain reaction. Then, the temperature is decreased until it reaches the annealing temperature which can vary depending on the set of primers used, their concentration, the probe
and its concentration (if used), and the cations
concentration. The main consideration when choosing the optimal annealing temperature is the melting temperature (Tm) of the primers and probes (if used). The annealing temperature chosen for a PCR depends directly on length and composition of the primers. This is the result of the difference of hydrogen bond
s between A-T (2 bonds) and G-C (3 bonds). An annealing temperature about 5 degrees below the lowest Tm of the pair of primers is usually used.
The final step of PCR amplification is DNA extension from the primers. This is done with thermostable Taq DNA polymerase
, usually at 72°C, the temperature at which the enzyme works optimally. The length of the incubation at each temperature, the temperature alterations, and the number of cycles are controlled by a programmable thermal cycler. The analysis of the PCR products depends on the type of PCR applied. If a conventional PCR is used, the PCR product is detected using agarose gel electrophoresis and ethidium bromide
(or other nucleic acid staining).
Conventional RT-PCR is a time-consuming technique with important limitations when compared to real-time PCR techniques. This, combined with the fact that ethidium bromide
has low sensitivity, yields results that are not always reliable. Moreover, there is an increased cross-contamination risk of the samples since detection of the PCR product requires the post-amplification processing of the samples. Furthermore, the specificity of the assay is mainly determined by the primers, which can give false-positive results. However, the most important issue concerning conventional RT-PCR is the fact that it is a semi- or even a low-quantitative technique, whereas the amplicon can be visualized only after the amplification ends.
Real-time RT-PCR
provides a method in which the amplicons can be visualized as the amplification progresses using a fluorescent reporter molecule. There are three major kinds of fluorescent reporters used in real time RT-PCR, which are general non-specific DNA Binding Dyes such as SYBR Green
I, TaqMan Probes
and Molecular Beacons
(including Scorpions).
The real-time PCR thermal cycler
has a fluorescence
detection threshold, below which it cannot discriminate the difference between an amplification generated signal and background noise. On the other hand, the fluorescence increases as the amplification progresses and the instrument performs data acquisition during the annealing step of each cycle. The number of amplicons
will reach the detection baseline after a specific cycle, which depends on the initial concentration of the target DNA sequence. The cycle at which the instrument can discriminate the amplification generated fluorescence
from the background noise is called the threshold cycle (Ct). The higher the initial DNA concentration, the lower its Ct will be.
analysis is used to study the RNA's gene expression further. 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 which 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 eukaryote
RT-PCR is commonly used in studying the genomes of virus
es whose genomes are composed of RNA, such as Influenzavirus A
and retrovirus
es like HIV
.
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....
(PCR), a laboratory technique commonly used in molecular biology
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...
to generate many copies of a 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...
sequence, a process termed "amplification". In RT-PCR, however, an RNA strand is first reverse transcribed
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...
into its 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...
complement (complementary DNA
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...
, or cDNA) using the enzyme 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...
, and the resulting cDNA is amplified using traditional PCR or real-time PCR
Real-time polymerase chain reaction
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction or kinetic polymerase chain reaction , is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule...
. Reverse transcription PCR is not to be confused with real-time polymerase chain reaction
Real-time polymerase chain reaction
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction or kinetic polymerase chain reaction , is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule...
(Q-PCR/qRT-PCR), which is also sometimes abbreviated as RT-PCR.
RT-PCR principles and procedure
RT-PCR utilizes a pair of primersPrimer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
, which are complementary to a defined sequence on each of the two strands of the cDNA. These primers
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
are then extended by a DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
and a copy of the strand is made after each cycle, leading to exponential amplification.
RT-PCR includes three major steps. The first step is reverse transcription (RT), in which RNA is reverse transcribed to cDNA using 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...
. This step is very important in order to perform PCR since DNA polymerase can act only on 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...
templates. The RT step can be performed either in the same tube with PCR (one-step PCR) or in a separate one (two-step PCR) using a temperature between 40°C and 50°C, depending on the properties of the reverse transcriptase used.
The next step involves the denaturation of the dsDNA at 95°C, so that the two strands separate and the primers can bind again at lower temperatures and begin a new chain reaction. Then, the temperature is decreased until it reaches the annealing temperature which can vary depending on the set of primers used, their concentration, the 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...
and its concentration (if used), and the cations
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
concentration. The main consideration when choosing the optimal annealing temperature is the melting temperature (Tm) of the primers and probes (if used). The annealing temperature chosen for a PCR depends directly on length and composition of the primers. This is the result of the difference of hydrogen bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
s between A-T (2 bonds) and G-C (3 bonds). An annealing temperature about 5 degrees below the lowest Tm of the pair of primers is usually used.
The final step of PCR amplification is DNA extension from the primers. This is done with thermostable Taq DNA 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...
, usually at 72°C, the temperature at which the enzyme works optimally. The length of the incubation at each temperature, the temperature alterations, and the number of cycles are controlled by a programmable thermal cycler. The analysis of the PCR products depends on the type of PCR applied. If a conventional PCR is used, the PCR product is detected using agarose gel electrophoresis and ethidium bromide
Ethidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...
(or other nucleic acid staining).
Conventional RT-PCR is a time-consuming technique with important limitations when compared to real-time PCR techniques. This, combined with the fact that ethidium bromide
Ethidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...
has low sensitivity, yields results that are not always reliable. Moreover, there is an increased cross-contamination risk of the samples since detection of the PCR product requires the post-amplification processing of the samples. Furthermore, the specificity of the assay is mainly determined by the primers, which can give false-positive results. However, the most important issue concerning conventional RT-PCR is the fact that it is a semi- or even a low-quantitative technique, whereas the amplicon can be visualized only after the amplification ends.
Real-time RT-PCR
Real-time polymerase chain reaction
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction or kinetic polymerase chain reaction , is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule...
provides a method in which the amplicons can be visualized as the amplification progresses using a fluorescent reporter molecule. There are three major kinds of fluorescent reporters used in real time RT-PCR, which are general non-specific DNA Binding 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...
I, TaqMan Probes
TaqMan
TaqMan probes are hydrolysis probes that are designed to increase the specificity of real-time PCR assays. The method was first reported in 1991 by researchers at Cetus Corporation, and the technology was subsequently developed by Roche Molecular Diagnostics for diagnostic assays and by Applied...
and Molecular Beacons
Molecular beacon
Molecular beacons are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. The terms more often used is molecular beacon probes. Molecular beacons are hairpin shaped molecules with an internally quenched fluorophore whose fluorescence...
(including Scorpions).
The real-time PCR thermal cycler
Thermal cycler
The thermal cycler is a laboratory apparatus used to amplify segments of DNA via the polymerase chain reaction process. The device has a thermal block with holes where tubes holding the PCR reaction mixtures can be inserted...
has a fluorescence
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...
detection threshold, below which it cannot discriminate the difference between an amplification generated signal and background noise. On the other hand, the fluorescence increases as the amplification progresses and the instrument performs data acquisition during the annealing step of each cycle. The number of amplicons
Amplicons
thumb|75px|PCR ThermocyclerAn amplicon is a piece of DNA formed as the product of natural or artificial amplification events. For example, it can be formed via polymerase chain reactions or ligase chain reactions , as well as by natural gene duplication.Artificial amplification can be used to...
will reach the detection baseline after a specific cycle, which depends on the initial concentration of the target DNA sequence. The cycle at which the instrument can discriminate the amplification generated fluorescence
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...
from the background noise is called the threshold cycle (Ct). The higher the initial DNA concentration, the lower its Ct will be.
Use of reverse transcription polymerase chain reaction
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. 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,...
analysis is used to study the RNA's gene expression further. 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 which 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 eukaryote
RT-PCR is commonly used in studying the genomes of virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es whose genomes are composed of RNA, such as Influenzavirus A
Influenzavirus A
Influenza A virus causes influenza in birds and some mammals and is the only species of Influenzavirus A. Influenzavirus A is a genus of the Orthomyxoviridae family of viruses. Strains of all subtypes of influenza A virus have been isolated from wild birds, although disease is uncommon...
and retrovirus
Retrovirus
A retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...
es like HIV
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
.