What is the full form of RT-PCR

RT-PCR: Real-Time Reverse Transcription-Polymerase Chain Reaction

RT-PCR was ascended to turn into the benchmark innovation for the location and the examination of RNA levels in light of multiple factors:

It doesn't need post-PCR handling.
We can estimate a wide reach (>107-overlap) of RNA overflow.
It gives an understanding of both subjective and quantitative data.

Due to its effortlessness, particularity and responsiveness, RT-PCR is utilized in many applications, from tests as straightforward as a measurement of yeast cells in wine to additional perplexing purposes as symptomatic devices for recognizing irresistible specialists, for example, the avian seasonal infection and SARS-CoV-2.

Since its presentation in 1977, Northern smudge has been utilized widely for RNA evaluation regardless of its weaknesses:

Tedious procedure.
Requires a huge amount of RNA for identification.
Quantitatively erroneous in the low overflow of RNA content.

However, since Kary Mullis developed PCR in 1983, RT PCR has since dislodged Northern smear as the strategy for decision for RNA recognition and quantification.

Real-Time Reverse transcription-polymerase chain reaction ( RT-PCR) is one method utilized in the labs with the usage of reverse transcription of RNA to DNA and the development of specified targets in DNA by operating Polymerase Chain Reaction (PCR). It is widely used to count the amount of specific RNA. Using a method called Real-Time PCR or quantitative Polymerase Chain Reaction (qPCR), measuring the amount of specific RNA is achievable by observing the development reaction using fluorescence called real-time PCR.

The combination of RT-PCR and qPCR are used extensively for quantifying viral RNA to observe the expression of genes for medical and pharmaceutical uses. Because of the close association between RT-PCR and qPCR, people tend to confuse that both are the same. This is wrong as the RT-PCR can be used without quantitative PCR for simple detection of RNA and to enable molecular cloning and sequencing. Alternatively, quantitative PCR measures the duplicate number in a specified part of RNA or DNA.

Nomenclature

Polymerase Chain Reaction is abbreviated as 'PCR'. 'qPCR' is used for the real-time polymerase chain reaction technique. RT-PCR and qPCR combined technique is abbreviated as 'qRT-PCR'. The combined RT-PCR plus qPCR method is called quantitative RT-PCR or real-time RT-PCR and is also called quantitative real-time RT-PCR. It has several abbreviations such as 'qRT-PCR', 'RT-qPCR', 'RRT-PCR' and 'RRT-PCR'.

History

In 1983, the technique RT-PCR was first introduced. The northern blot technique, introduced in 1977, was widely used for quantifying RNA regardless of the limitations. It should have a huge amount of RNA for detection purposes.

For RNA detection and quantification, RT-PCR replaced the Northern Blot technique when it was introduced in 1983 by Kary Mullis and was assigned to Cetus Corporation. Patents covered Taq polymerase enzyme too, but a dispute is going on in many courts between Promega and Roche. The final expiration for the Taq polymerase enzyme patent claim happened on 28 March 2005. There have been huge controversies and disputes that happened during the patent assignment. A lawsuit which was of high profile also been filed against the patent issue. In 1992, a pharmaceutical company from swiss finally bought the patents, and it got expired in 2017.

Because of its specificity, simplicity and sensitivity, it is widely used for the simple process of quantification of yeast cells in wine, and for most complex uses such as the diagnosis of avian flu virus, SARS-CoV-2 and COVID -19.

RT-PCR method is recorded as the standard method for detecting as well as comparing various RNA levels taking into count many reasons like :

It does not require post PCR processing
Wide range of RNA abundance can be determined (>107-fold)
This technique provides analysis of both qualitative and quantitative data.

Variations

There are many variations discovered in RT-PCR.

Allele-specific PCR
Assembly PCR
Asymmetric PCR
Convective PCR
Dial out PCR
Digital PCR
Helicase dependent amplification
Hot start PCR
In silico PCR
Intersequence specific PCR
Inverse PCR
Ligation mediated PCR
Methylation-specific PCR
Miniprimer PCR
Multiplex PCR
Multiplex ligation-dependent probe amplification
Multiplex PCR
Nanoparticle Assisted PCR
Nested PCR
Overlap extension PCR
PAN AC
Quantitative PCR
Reverse Complement PCR
Reverse Complement PCR
Reverse Transcription PCR
RNase H Dependent PCR
Single Specific Primer PCR
Solid Phase PCR
Suicide PCR
Thermal asymmetric interlaced PCR
Touchdown PCR
Universal Fast Walking PCR

Application of RT-PCR

The RT-PCR technique is extensively useful in diagnosing the diseases caused by genetics, with a quarter quantification, as the measure of gene expression by concluding the huge finding of specified, unlike RNA molecules in a single tissue or cell. The ascending development through reverse transcription-polymerase chain reaction aids for a highly sensitive method with fewer duplicate RNA molecules that can be observed. RT-CPR is also used in forensic applications such as recognizing fingerprints at crime scenes within minutes.

The major advantage of this technique is that instead of filtering out the results from the DNA database, we can rule out the database results and conclude the results from RNA. In research applications, PCR is used for DNA cloning. It is also used to find DNA from ancient bones and Egyptian mummy brains. If the DNA is degraded much, it can be identified at the early stages. Scientists are working on using RT-PCR in detecting cancer to assist in improving prognosis and supervising their records to therapy. Transmitting tumour cells gives different mRNA transcripts according to the cancer type.

The motto is to find out which mRNA transcripts are the best biomarkers for a specific cancer cell type and then inspect its equation levels with RT-PCR. 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

Research Method

The RT-PCR technique is the most common method used for research in determining gene expression. Lin et al. used the qRT-PCR method to calculate the equation of Gal genes in yeast cells. Lin et al. created a mutation in a specific protein which he suspected to take part in regulating Gal genes. By the hypothesis, this mutation is the reason for selectively eliminating Gal expression.

To determine the accuracy of this hypothesis, Lin et al. used the technique of qRT-PCR to check the gene expression levels of yeast cells in which this mutation is present. By the qRT-PCR, researchers concluded that the part of this particular protein reduced Gal expression. The scientists later utilized Northern blot analysis to determine the RNA's gene equation.

RT-PCR effect on COVID-19 Virus

When the covid-19 hit all our lives in 2020, we all know how the situation was. Then, the United States organization in Food and Agriculture has done many pieces of research on this real-time reverse transcription-polymerase chain reaction. RT-PCR is used for tracking, studying and detecting coronavirus. It is related to nuclear technology. It has been used to detect viruses like Zika virus and Ebola virus in the past. People wanted to adopt the technique in the times of the covid-19 virus.

Initially, researchers collected swabs or samples from the COVID-19 patient's noses and throats. The samples were then treated and removed of substances like the proteins and fats. This extraction will turn out into a sample given person generic material. The extract will show the RNA of the virus too. And that's how RT-PCR can work with the COVID-19 virus. Researchers have been working on this method at the time of covid and over the past twenty years. These works are mostly held on the VETLAB network of veterinary diagnostic labs. This technology has also detected some major animal diseases that can infect humans.

Uses of RT-PCR

RT-PCR can instantly detect diseases like cancer, infections, and gene abnormalities. These all can be detected only with the help of nucleic acid. This method has helped with many contagious diseases. It has gradually become a tool for detecting diseases. Microbiologists use quantitative PCR to eliminate food spoilage, food safety and fermentation.

It is also used for detecting microbes in water. Quantitative PCR can also be called qPCR, which is used to amplify genes in DNA from environmental samples. RT-PCR is also used in the agricultural industry for producing germ-free seedlings, which can eventually remove economic losses.

Advantages

The medical industry is told that RT-PCR is simple to understand and use. It is a practical and powerful tool.

Drawbacks

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. One of the biggest drawbacks to this RT-PCR is the information prior is needed to face the upcoming consequence. Like the other error-prone systems, RT-PCR is also prone to errors. The other limitation is that even if a smaller amount of DNA is present along with RNA while testing, it gets contaminated, and results get misled. So to avoid this sort of misleading result, researchers choose to create separate reagent rooms for RNA analysis. Sometimes, humic acids from environmental samples may also lead to erroneous results. It depends upon the total hygiene of the laboratory.