In the reverse transcription (RT)-PCR, the cDNA synthesised from RNA using the reverse transcriptase enzyme and is amplified and quantified for gene expression studies.
In other words, we can define it as,
"The amount of the RNA present into the sample is quantified by using the fluorescent dye or probe by synthesising cDNA from RNA using the reverse transcriptase enzyme."
In genomic research, the PCR has unmatched applications. The PCR is utilised in almost every genetic research tool. Because it helps in producing a large amount of specific gene segment or gene of our interest.
In the conventional PCR, the DNA is synthesized with the help of the Taq DNA polymerase (in replication it is just a DNA polymerase).
The Taq DNA recognizes the binding site or substrate ( the ssDNA and primer binding junction) and adds dNTPs to the growing DNA strand.
Any types of DNA source whether it is a human gDNA, plasmid DNA or viral DNA can be amplified into PCR using the Taq DNA polymerase.
But the problem of amplification is originated while doing the research on HCV, HIV or influenza, all these viruses are RNA virus. The Taq DNA polymerase can not synthesize the RNA.
However, during the 90s, the northern blot method for RNA determination is used for a long time but the method is tedious, time-consuming and unreliable.
The problem of amplification of RNA is solved after the discovery of the reverse transcriptase enzyme.
In this article, we are going to understand another important type of PCR method, reverse transcription PCR in which instead of DNA the starting material is RNA.
Overview of reverse transcription PCR:
The process of central dogma is the lifeline of any organism on earth.
The process of replication, transcription and translation is called central dogma. In the replication, the DNA is doubled, in the transcription, the mRNA is formed from the DNA and from that mRNA the long chain of the amino acid is synthesised, in translation.
Take a look at the process of replication: General process of DNA replication
The mechanism of central dogma is almost the same in all organisms (except RNA viruses).
The reverse transcription PCR also called as a quantitative PCR or quantitative RT-PCR determines the expression of total RNA present into the sample by complementary DNA sequence.
For characterization of particular mutation or disease, we are using the DNA as a sample then what is the need for RNA.
The answer is here,
The total DNA contains coding and non-coding DNA. Even if we target a particular gene, the gene contains coding exons, non-coding introns and promoter.
On the other side, once the amino acid chain forms the protein is synthesised only from the coding region of the gene viz by the exon only.
The amount of the protein present in particular tissue is directly proportional to the amount of mRNA formed from a gene.
Therefore, if we calculate the total amount of mRNA, ultimately we are calculating the amount of the gene which encodes a particular protein.
The figure below is the graphical representation of the central dogma process.
The starting material for the RT-PCR is RNA, hence we have to extract RNA instead of DNA. Oligo (dT) primers, random primers and sequence-specific primers are three types of primers can be used in the quantitative RT-PCR. However, which primer set are used depends on the requirement of the assay.
The entire technique of reverse transcription PCR is depended on the reverse transcriptase enzyme.
Common abbreviations for reverse transcription PCR:
|RT-PCR||Reverse transcription PCR|
|qRT-PCR||Quantitative-Reverse transcription PCR|
|RT-qPCR||Reverse transcription-quantitative PCR|
Reverse transcriptase enzyme:
The reverse transcriptase enzyme is discovered by Howard Temin and Satoshi Mizutani however, the enzyme is first isolated in the year 1970 by David Baltimore. The enzyme is predominantly present into the retroviruses having the RNA as genetic material.
In the retrovirus,
Instead of synthesising RNA, the DNA (called a complementary DNA) is synthesised from the RNA. Once the DNA is formed, it is integrated into the host genome and replicate, transcribe and translate along with the host genome and ultimately, the viral protein formed.
The enzyme is RNA dependent-DNA polymerase or RNA directed DNA-polymerase present into the retroviruses. The retrovirus contains only three genes, ‘gag‘, ‘pol‘ and ‘env‘. The entire process of retroviral replication called reverse transcription.
This reverse transcriptase enzyme is isolated from the bacteria and used in the PCR.
The principle of reverse transcription PCR:
With the help of the reverse transcriptase enzyme, the cDNA is synthesised using the target RNA. The RT-PCR is broadly divided into two steps, first is the reverse transcription and second is the amplification and quantification. Depending upon that the RT- qPCR can be performed by two methods:
- One-step RT-PCR
- Two-step RT-PCR
Here in the one-step RT-PCR both the reverse transcription and the amplification can be performed in the single reaction, hence in the single tube.
The setup of one-step reverse transcription PCR is simple and easy. Also, the accuracy and the specificity of reaction is higher, therefore, it is the best choice for high throughput screening and for repeated quantification assays.
Additionally, the reaction is completed in the single tube so the chance of the contamination is very less in comparison with two-step RT-PCR.
Although the reaction is fast and reproducible, it detects less template per sample. Further, the reaction conditions are compromised because of the use of so many chemicals in one reaction.
The major limitation of the one-step RT-PCR is the stock of cDNA. Here, the cDNA is directly synthesized into the reaction tube, therefore, we can not run two reactions separately for one sample type because the cDNA stock cannot be stored for further use.
The requirement of a template is higher as it quantifies the template from the initial as well as from the exponential phase.
The chance of non-specific bindings and primer dimers formation are also higher in this method because one-step RT PCR utilizes only sequence-specific primers.
See the figure below,
Contrary to the one-step method, in the two-step RT-PCR the reverse transcription and amplification are performed in two different reaction tubes.
The reaction conditions are different for both the reaction.
In the first reaction, the cDNA is synthesised from the RNA for that reverse transcriptase, buffer, dNTPs reaction mixture, oligo (dT) primers and random primers are used.
After the reaction complete, we have the cDNA stock for the second set of reaction. Here the two-step RT-PCR facilitate one additional benefit over one-step RT-PCR, we can store this cDNA for further reactions.
In the second reaction, instead of reverse transcriptase, the normal DNA polymerase along with other PCR essentials are added into the tube.
The cDNA is now amplified and quantified into the reaction.
In the two-step RT-PCR, we are using three sets of primers, one set of oligo(dT) primers, one set of random primers and sequence-specific primers hence the specificity of the reaction is higher as compared with one-step RT-PCR reaction.
The method is advantageous for the smaller amount of sample. Nevertheless, due to the additional step, the chance of the contamination and reaction failure is higher in the two-step RT-PCR reaction.
Although the accuracy and specificity are higher in this method, it is time-consuming consequently it is not used for high throughput screening.
The two-step RT-PCR method required more expertise and additional optimization.
This two types of methods are used for the gene expression RT-qPCR. The method is shown in the figure below,
The procedure of reverse transcription PCR:
The procedure of RT-qPCR completed in the following steps,
- Sample preparation
- Selection of primers
- Reaction preparation
- RT-PCR cyclic condition
- Strand synthesis
Instead of DNA, RNA is extracted for the RT-PCR. For extracting the RNA use ready to use RNA extraction kits, it performs better and the yield of the extraction is even good.
Now, the question strikes in mind that we are encountering the gene expression, then why to extract total RNA instead of the only mRNA?
We can extract the only mRNA for the RT-qPCR but the quantity might not be sufficient for the experiment, also, additional purification and mRNA separation steps are required.
A good quantity of RNA is obtained by extracting the total RNA.
So use total RNA instead of the only mRNA.
Selection of primers:
In the next step select the primer for the experiments.
Three types of primers can be used in the reverse transcription PCR.
- Random primers
- Oligo(dT) primers
- Sequence-specific primer
1. Random primers:
Random primers are short single-stranded sequences of hexamers or octamers. The random primer binds at the complementary random location on the RNA. It can bind to many types of RNA (tRNA, rRNA or mRNA) and synthesised the cDNA.
It is used in the RT-PCR specifically for the templates having huge secondary structure. The yield of cDNA synthesis is very high by using the random hexamers, However, it can produce truncated cDNA.
Random primers cannot bind to the poly-A tail of RNA, therefore, it is not preferred for the longer eukaryotic poly-A tail containing RNA. Although it is a good choice for smaller RNA, tRNA, rRNA or prokaryotic RNA.
The random primers are not preferred for longer full-length RNA because it can synthesise shorted cDNAs efficiently and if the concentration of random primers is higher, the fragments of cDNA are even smaller.
2 to 5 μM concentration of random primers are enough for RT-PCR.
See the figure, it shows the location of random primer binding on the RNA.
2.Oligo (dT) primers:
The mRNA contains a chain of the Poly-A tail at the end of the mRNA. The oligo (dT) primers bind to the tail of the mRNA ensure the synthesis of full-length mRNA into cDNA. The oligo (dT) primers play a critical role in the low quantity RNA sample.
It actually synthesises the whole stretch of the mRNA.
The oligo (dT) primers are 12 to 18 nucleotide long single-stranded DNA which contains one additional nucleotide at the 3′ end to anchor the binding.
The anchored (dT) primers prevent the primer slippage and the denaturation of primer from the poly-A tail.
However, the oligo (dT) primers can not synthesise RNA other than mRNA because the tRNAs, rRNAs and other degraded and micro RNA does not have the poly-A tail.
2 to 4 μM concentration of oligo (dT) primers are enough for RT-PCR.
See, the figure below which shows the binding of oligo (dT) primers to the mRNA.
The sequence-specific primers are complementary to the sequence of our interest. The sequence-specific primers are used in the one-step RT PCR because it required more amount of RNA.
It synthesises cDNA only of our interest from the RNA hence large amount of template RNA is required for one-step PCR.
Due to the higher specificity of sequence-specific primers, it can synthesis only specific sequence from the total RNA.
See the figure below,
The sequence-specific primers synthesise only certain regions from the RNA, hence less amount is recommended to achieve success in the reaction. 0.5 to 1.5 μM concentration is enough for the RT-qPCR.
Note: If sequence-specific primers and oligo (dT) primers both are used in a single reaction, use only 1μM each primer.
The figure above shows the specificity of the sequence-specific primers, as it can only bind to the mRNA and cannot binds to gDNA.
Components used in the RT-qPCR:
The selection of components for the reverse transcription PCR is as crucial as selecting a temperature condition but don’t worry about it, the ready to use reverse transcription PCR kit contains all the ingredients into the reaction buffer and reaction mixture.
The major components are,
- DNA primers
Read more on PCR primers: PCR primer design guidelines
Read more on dNTPs: The Function of dNTPs in PCR reaction
- Reverse transcriptase enzyme with RNase activity
- RNase H (if the reverse transcriptase does not have it)
- DNA polymerase
Read more on Taq DNA polymerase: Function of Taq DNA polymerase in PCR
- RT-qPCR buffer with RNase inhibitors and PCR enhancers
Read more on inhibitors: Effect of PCR inhibitors on PCR amplification
- DEPC treated nuclease-free water
- DNA ligase
Temperature conditions of RT-qPCR:
Here, the denaturation step is not required. The PCR reaction starts with the primer annealing. At the first stage the primer annealed to the template RNA, once it annealed, the reaction is placed for cooling at 4°C for proper binding.
After that, the strand synthesis is started in stage two followed by the deactivation of the enzyme in stage three.
Note: The temperature in each stage of the reaction may vary depending on the length of the primers, composition of primers, the types of enzyme used in the reaction and the length of the amplicon.
First-strand and second-strand cDNA synthesis:
Synthesis of cDNA is a crucial step in the entire RT-PCR protocol. The cDNA synthesis is completed into a two-step, first-strand cDNA synthesis and second-strand DNA synthesis.
In the beginning, the reverse transcriptase enzyme synthesised the cDNA from the single-stranded RNA and is called first-strand cDNA synthesis. See step 1 in the figure below.
The reverse transcriptase enzyme does have the RNase activity that cleaves the RNA from the RNA-cDNA hybrid into smaller fragments.
If the reverse transcriptase enzyme does not have the RNase activity, add E.coli RNase H separately into the reaction.
Remember, do not use a higher concentration of RNase H because it cleaves the longer RNA template prematurely.
Now, the nicks generated by the RNase H activity is filled by the DNA polymerase.
A special types of DNA polymerases such as DNA polymerase I and T7 DNA polymerase are used to fill the nick.
The special types of DNA polymerase synthesise DNA from 5′ to 3′ direction by removing the RNA fragments in 5′ to 3′ exonuclease activity.
After the synthesis of the new DNA fragment, the nicks between adjacent DNA are ligated by the ligase enzyme. Here, use E.coli DNA ligase instead of T4 DNA ligase.
The second strand is now synthesised from the RNA.
The mechanism of strand synthesis is explained into the figure below,
Note: The wild types Moloney murine leukaemia virus reverse transcriptase and Avian myeloblastosis virus reverse transcriptase is a good choice for RT-qPCR because both have adequate RNase H activity.
Applications of reverse transcription PCR:
- Tissue-specific gene expression can be determined by the reverse transcription PCR.
- The RT-qPCR is applicable in monitoring the prognosis and therapy response in the cancer patients.
- Also, the mutant gene and its expression in particular tissue can be determined by using this method.
- It is used in the gene insertion studies.
- The inherited disease can also be diagnosed by using RT-PCR.
Advantages of reverse transcription PCR:
- The method can do quantitative as well as qualitative analysis.
- A small amount of RNA can be used for gene expression studies.
- Post PCR processing such as agarose gel electrophoresis is not required here.
- The method is simple, easy to use, rapid and cost-effective.
- Also, the specificity and the sensitivity of the reverse transcription PCR is enormous.
Disadvantages of reverse transcription PCR:
- The method is extremely sensitive, even a small amount of DNA contamination can lead to false results.
- The method is restricted for some of the assays as higher expertise and experimentation are required to develop new assays.
- What is a multiplex PCR?
- Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications
The reverse transcription PCR or RT-qPCR or qRT- PCR is a gold standard method for HIV and HPV detection.
Conclusively, we can say the reverse transcription PCR can be a good choice for gene expression studies nevertheless, the sequence of DNA cannot be determined by RT-qPCR.