Rt -Pcr

Sutrisha Kundu

7th September, 2020

Polymerase Chain Reaction (PCR), a technique used to amplify a segment of DNA by producing multiple copies of it from an initially small sample, is one of the most useful techniques in molecular biology.

 

Now, what if the sample is RNA and not DNA? What if we want to amplify the genomic material of an RNA virus? Fortunately, Science has an answer to all questions. 

 

Scientists developed RT PCR, where RT stands for Reverse Transcription, after the discovery of the reverse transcriptase enzyme. Using this enzyme, cDNA can easily be constructed from the RNA sample, and then PCR is conducted using the DNA obtained.

 

Reverse Transcription PCR is often used as a quantitative form of PCR as well because it measures gene expression by amplifying cDNA reverse transcribed from RNA. The total genomic DNA has coding as well as non-coding sequences. Coding sequences or genes can be further classified into exons and introns. During translation, the amino acid is formed only from the exon part of the DNA. Hence, during transcription, only the coding sequence, that is exons, forms the transcript or mRNA. Therefore, by quantifying the RNA, the amount of a gene present in a cell can be measured or determined.

Procedure:

The whole process of RT PCR includes the following steps:

  • Sample preparation

  • Selection of primers

  • RT-PCR cyclic condition

  • Strand synthesis

  • PCR amplification

 

Sample preparation – RNA is extracted using an RNA extraction kit. Care must be taken while extraction because RNase present on every possible surface in the lab will cleave the RNA. 

 

Selection of primers – Three different kinds of primers can be used for RT PCR depending on the required condition.

  1. Random primers – Smaller fragments of RNA are amplified using random primers. It is used in the RT-PCR particularly for templates having a secondary structure.

  2. Oligo (dT) primers – The oligo (dT) primers are specially designed to amplify the mRNA. It is a 12 to 18 nucleotide long single-stranded DNA which contains one additional nucleotide at the 3′ end to anchor the binding.

  3. Sequence-specific primers – The sequence-specific primers are commonly utilized in one-step RT-PCR to amplify a gene of interest. Due to its high sequence specificity, it is preferred more in gene expression studies.

 

RT-PCR cyclic condition – Since RNAs are single-stranded, there is no need for any denaturation step. The PCR reaction starts directly with the primer annealing. Once the primer binds properly to the RNA, new strand synthesis occurs which is then followed by enzyme deactivation in step three, as shown in the figure below.

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Strand synthesis – The synthesis of cDNA is considered to be the most crucial step in the entire RT-PCR protocol. The synthesis is completed into two steps.

In the first step, the reverse transcriptase enzyme amplifies the cDNA from the single-stranded RNA.

The RNA DNA hybrid is then cleaved by the RNase H enzyme. The nicks generated are then filled up by special DNA polymerases. The same DNA polymerase then synthesizes the second DNA strand from 5′ to 3′ direction by removing the RNA fragments using its exonuclease activity, just like lagging strand synthesis during replication. Finally, the nicks are ligated by the ligase enzyme, as shown in the picture given below.

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PCR amplification – Once the cDNA is obtained, the PCR cycle is run to obtain multiple copies using specific PCR primers for the desired regions. One must always keep in mind that this RT PCR amplification does not tell us about the genomic content of the cells but rather the transcription level of specific genes. 

 

 

This whole process of RT PCR can be done in either of the following ways:

a) One-step RT PCR

b) Two-step RT PCR

 

One-step RT PCR, both reverse transcription and amplification are performed in a single tube. It is widely used in repeat quantification assays and high throughput screening due to its high accuracy, easy to use, and simple set up.

3.png

On the other hand, in the two-step RT-PCR, reverse transcription and amplification are performed in two separate reaction tubes. Both reactions have different conditions and ingredients used in it. In the first reaction, cDNA is synthesized and in the second step, the cDNA is amplified. One of its greatest advantages is that the cDNA can be stored for other future uses.

4.png

Two-step RT-PCR

The use of RT PCR has increased rapidly in recent times when it is being used to detect the presence of COVID-19 virus in human beings.

A sample is collected from the parts of the body where the COVID-19 virus gathers, such as a person’s nose or throat. The RNA (a mix of the patient’s RNA and the viral RNA, if present) is extracted from the sample. After the RNA is reverse transcribed into the DNA, PCR cycles are run using fluorescent markers and specific DNA primers, complementary to the specific parts of the transcribed viral DNA. By measuring the amount of fluorescence after each cycle, the presence of the virus can be verified.

The different kinds of PCR are gradually gaining more and more important in fields like molecular biology, genetics, and genome research. It is one of the most suitable ways to detect disease-causing pathogens. Here lies the importance of RT PCR which proves to be the most convenient and simplest way to detect RNA viruses, one of the deadliest diseases causing pathogens because of the more frequent mutations occurring in RNAs.



 

References:

[1] Reverse Transcription setup – www.thermofisher.com

[2] Reverse Transcription Polymerase Chain Reaction – www.sciencedirect.com

[3] Reverse Transcription PCR: Principle, Procedure, Applications, and Limitations – geneticeducation.co.in

[4] Quantitative RT – PCR An overview of current methodologies. Joyce C (2002). 

[5] How is the COVID-19 Virus Detected using Real-Time RT-PCR? - iaea.org

[6] Applications of Reverse Transcription PCR – www.ncbi.nlm.nih.gov

 

 

                                                                                  

About the Author

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Sutrisha is from St. Xavier's College, Kolkata, currently studying in 3rd year of Integrated MSc in Biotechnology. She is engaged in an ongoing project titled "Rice Hisk Ash."