Reverse transcription of the ribonucleic acid: the first step in RT-PCR assay.
Similar to the RT-PCR workflow, RNA is first converted to cDNA, which is then amplified by PCR. The main difference, however, is that levels of amplified cDNA . In genetics, complementary DNA (cDNA) is DNA synthesized from a single- stranded RNA (e.g., messenger RNA (mRNA) or microRNA) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in prokaryotes. Supreme Court ruled in the case of Association for Molecular Pathology v. The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA.
Copyright Genetics Society of America Reverse Transcriptase Reverse transcriptase is the replication enzyme of retroviruses. Because reverse transcriptase is essential for retroviruses such as HIV -1 the virus that causes AIDSit is the target of many antiretroviral therapeutics. Reverse transcriptase is also a molecular tool used in the cloning of genes and the analysis of gene expression. Discovery Retroviruses were originally known as RNA tumor viruses because they have RNA, not DNA, genomes, and because they were the first viruses recognized to cause certain cancers in animals.
At the middle of the twentieth century, Howard Temin was interested in understanding how RNA tumor viruses cause cancer. One finding that interested him was the genetic-like stability of the uncontrolled cell growth caused by these viruses.
It was known then that certain bacterial viruses, called phages, could integrate their DNA into their hosts' chromosomes and persist as stable genetic elements known as prophages. By analogy, Temin proposed the provirus hypothesis, which suggests that RNA tumor viruses can cause permanent alterations to cells by integrating into host chromosomes.
However, reverse transcription was at odds with the then-popular central dogma of molecular biologyadvanced by Francis Crick, which maintained that genetic information flowed unidirectionally from DNA to RNA to protein. Because his proposal of a reverse flow of genetic information from RNA to DNA seemed heretical, and because the experimental techniques needed to test this idea were not yet developed, Temin and his hypothesis were rebuffed for many years.
The biochemical proof for reverse transcription finally arrived in when two separate research teams, one led by Temin and the other by David Baltimoresimultaneously discovered the elusive RNA-copying DNA polymerase in purified virions.
In Temin and Baltimore shared the Nobel Prize in physiology or medicine for their discovery of reverse transcriptase. Laboratory Uses of Reverse Transcriptase Reverse transcriptase went on to play a critical role in the molecular revolution of the late s and s, especially in the fields of gene discovery and biotechnology. The cDNA can then be expressed in a model organism such as Escherichia coliand the protein it codes for can then be made in abundance.
The cloning of cDNA was instrumental to gene discovery in the later part of the twentieth century. Using cDNA copies of genes is necessary when bacteria are used to produce human protein-based pharmaceuticals. This is because bacteria lack the machinery necessary to recognize unspliced genes, but bacteria can use cDNAs to direct the synthesis of human or other higher organism proteins.
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Even though the human genome sequence was reported incopying RNAs with reverse transcriptase remains important. 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.
It is important for the design of the synthetic RNA be identical in sequence but slightly shorter than the target RNA for accurate results. 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. 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.
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. All of these probes allow the detection of PCR products by generating a fluorescent signal. 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.
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.
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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. 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.
- Basic Principles of RT-qPCR
- Reverse transcription of the ribonucleic acid: the first step in RT-PCR assay.
- Reverse transcription polymerase chain reaction
The Scorpion probes, like Molecular Beacon, 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. 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.
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.