“In the reverse transcriptase PCR, the reverse transcriptase constructs cDNA from the RNA in a cyclic temperature PCR reaction.”
PCR generates copies of DNA. Conventional PCR. Conventional PCR relies on the activity of Taq DNA polymerase having the power to synthesize DNA in vitro. PCR stands for Polymerase chain reaction, we all know.
Put simply, in a temperature-dependent reaction, the Taq DNA polymerase amplifies the template DNA using dNTPs. It uses the target DNA as substrate and using reaction buffers and primers it makes new DNA strands in vitro.
It helps in various genetics and genomic research and is applied in other fields such as microbiology, biotechnology, environmental science and forensic science.
Read more: What is Genetics?- Definition, History, Applications And Branches.
A variant of PCR known as qPCR quantifies genes or DNA present in the reaction, though it relies on a different setup. Reverse transcription or reverse transcriptase PCR is a type of quantitative PCR, applied to study gene expression and RNA viruses.
As retrovirus and other related viruses have RNA as their genetic material, their amplification and quantification are not possible using conventional Taq DNA polymerase. The reason is that Taq can only synthesize DNA not RNA.
And that’s why we need a special type setup and reagents in order to study the RNA, I will discuss it in this post.
During the ’90s Northern blotting was used to study the RNA but the method was tedious, time-consuming, and unreliable. If we somehow manage to convert the RNA into DNA; our task becomes easy.
After the discovery of the reverse transcriptase enzyme the problem was solved. The enzyme can synthesize DNA from RNA, revert back, and the new field of PCR technique evolved, the reverse transcription PCR.
The present blog post comprises information on reverse transcription PCR, its principle, process, advantages, limitations and applications. We will also thoroughly understand the mechanism and concept.
I hope this article will add more value to your PCR knowledge and help you in understanding the topic more.
Stay tuned,
What is reverse transcription PCR?
A PCR variant, Reverse Transcription PCR can synthesize DNA from the RNA abbreviated as RT-PCR, reverse PCR, qRT-PCR.
Definition:
We can define the process by either theoretical concepts or practical applications. Here are both.
Theoretical definition:
A type of PCR applied to convert the DNA from RNA using the reverse transcriptase enzyme is known as reverse transcription PCR.
Practical definition:
A type of PCR capable of quantifying the amount of RNA present in a sample using either fluorescent dye or probe by synthesizing cDNA from the RNA by reverse transcriptase enzyme, the process is known as reverse transcription PCR.
Overview and concept:
Replication transcription and translation are commonly known as the “central dogma of a cell” has importance for us. Replication doubles DNA, transcription forms mRNA, and translation forms protein or a chain of amino acids.
Read more:
The usual process in nature is synthesizing RNA from the DNA (the transcription). However several viruses known as retrovirus can synthesize DNA from the RNA by a special type of polymerase.
That polymerase is a key play in the present technique.
To study mutations or genotyping, we are using DNA then why do we need RNA quantification? Here is the answer,
Total genomic DNA has coding and non-coding sequences, coding sequences are known as genes that have exons and introns. During translation, the amino acid can be formed only from coding regions viz from the exons. So during transcription, only the coding sequence- exons of a gene forms a transcript or mRNA.
Therefore, by quantifying the mRNA, the amount of a gene present in a cell can be measured or determined. Although we can’t directly use RNA. We have to first convert it into DNA. This is the whole fundamental of gene expression studies.
Reverse transcription PCR, often known as quantitative PCR of quantitative RT-PCR measures gene expression by amplifying cDNA reverse transcribed from RNA. The figure below is the graphical representation of the central dogma process.
Common abbreviations for reverse transcription PCR:
| RT-PCR | Reverse transcription PCR |
| qRT-PCR | Quantitative-Reverse transcription PCR |
| RT-qPCR | Reverse transcription-quantitative PCR |
| qPCR | Quantitative PCR |
Reverse transcriptase enzyme:
Howard Temin and Satoshi Mizutani had discovered reverse transcriptase enzyme, although it was first isolated by David Baltimore in 1970. It is found in retroviruses and other RNA-containing viruses.
In the retrovirus, the enzyme forms DNA from its RNA which inserts into the host genome at where it is replicated, transcribed, and translated and forms viral proteins.
The enzyme is a type of RNA-dependent-DNA polymerase or RNA-directed DNA-polymerase. The retrovirus has only three genes, ‘gag‘, ‘pol‘, and ‘env‘ that forms polymerase, envelope, and other proteins of retrovirus thus the entire mechanism is known as reverse transcription.
RNA, set of primer, set of probes, dNTPs, reverse transcriptase, and PCR buffer are key ingredients of the assay. We have to first extract RNA instead of DNA. Next, the set of primers amplify the selected gene.
Oligo (dT) primers, random primers, and sequence-specific primers are three types of primers commonly used here. However, which primer is applied, depends on the type of RT-PCR assay.
Reverse transcriptase is a key player here, the overall success of the assay depends upon it.
Principle of reverse transcription PCR
The reverse transcriptase enzyme synthesizes the cDNA from the RNA. The process has two steps; the reverse transcription process and the second amplification and quantification.
A PCR makes the cDNA using the enzyme, dNTP mix, PCR reaction buffer, template RNA and quantifies it by adding probe or dye mixture to it. The process is divided broadly into two types, depending upon the RT- qPCR we perform.
One-step RT-PCR
Two-step RT-PCR
One-step RT-PCR:
In a single tube or single reaction, reverse transcription and amplification are performed (therefore it is named as one-step RT-PCR). The assay has more accuracy, specificity, easy handling approach and simple setup. Hence have applications in repeat quantification and high throughput screening.
In comparison to two-step PCR, it has less chances of reaction failure and contamination as the reaction only has a single step. In addition, it’s a fast, reproducible and cheaper process. Although it has a few shortcomings too,
Using many chemicals and reagents in a single tube can compromise reaction conditions, sometimes. It can detect less templates per reaction (or sample).
We can’t store the cDNA here as both cDNA synthesis, amplification and quantification occur simultaneously in a single reaction. It can’t be used in further reactions.
Also, we need more starting material or template to get good results as the template amplifies in both initial and exponential phases.
It also has more chances of reaction failure, primer-dimer and non-specific bindings because of the use of sequence-specific primers in a single reaction. The entire process of one-step PCR is explained in the figure below,
Two-step RT-PCR:
Contrary to the one-step method, two different reactions, in two different tubes, for reverse transcription and amplification, have been prepared. That is why this variation is known as two-step RT-PCR. Notably, both reactions have different conditions and ingredients used.
The first reaction uses reverse transcriptase enzyme, dNTPs, reaction buffer, oligo-(dT) primers, and random primers to build a cDNA. After completion of the reaction, we get cDNA stock.
We can either store the stock cDNA at adequate conditions or can use it directly for gene expression study. The second step uses the cDNA as a template to quantify the mRNA.
In the second reaction, along with all PCR reagents, the Taq DNA polymerase completes the reaction and quantification using a dye or probe. Here we do not need a reverse transcriptase. The second reaction quantifies our cDNA (formed from the first step).
One of the biggest advantages of the two-step method is that we can store the cDNA reverse transcribed from the RNA.
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 henceforth it has high specificity compared to the one-step method.
The present method performs so well for a smaller number of samples nevertheless, the use of additional steps makes it more prone to reaction failure and contamination. Thus the two-step RT-PCR isn’t advisable, ideally.
Since the method is specific, accurate, and more reliable than one-step methods, still, it can’t be utilized in high throughput screening. The reason is that it is time-consuming, prone to contamination and costlier (as more reagents are required).
Last but not the least, from my personal experience I can say, broad experience and expertise are required to perform two-step PCR. Usually, it is used in viral gene expression studies. The graphical illustration of the process is shown in the figure below,
Steps and Procedure of reverse transcription PCR:
The procedure of RT-qPCR is completed in the following steps,
- Sample preparation
- Selection of primers
- Reaction preparation
- RT-PCR cyclic condition
- Strand synthesis
Sample preparation:
We need to extract DNA instead of DNA to perform RT-PCR. For extracting the RNA use ready-to-use RNA extraction kits, it performs better and gives more yield.
We have to extract RNA, not DNA. Care must be taken while extraction as RNase is present on every possible surface in a lab. RNase is an enzyme that cleaves RNA. Use a ready-to-use RNA extraction kit to avoid problems in extraction.
So use total RNA instead of the only mRNA.
Selection of primers:
Designing and selecting primers have immense importance in PCR or in any PCR. The primer designed should follow all the criteria of standard primers. The assay needs three types of primers.
Random primers
Oligo(dT) primers
Sequence-specific primer
2. 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 synthesizes the cDNA.
It has importance particularly for the templates having a huge secondary structure. Random hexamer provides high yield cDNA. nonetheless, it can produce truncated cDNA.
The random primers work finely for prokaryotic RNA, tRNA, rRNA, and other smaller RNAs. But as it can’t bind to a poly-A tail, it is less preferred for eukaryotic RNA amplification.
Random primers can easily amplify smaller RNA fragments. 2 to 5 μM concentration of random primers is enough for RT-PCR. Higher concentration can make the reaction ineffective. The hexamer bindings on RNA are shown in the figure below.
2.Oligo (dT) primers:
The oligo (dT) primers are specially designed to amplify the mRNA. As we know that the mRNA has a poly-A tail, the oligo (dT) primers only bind to the poly-A tail of mRNA. Hence it can amplify the entire mDNA into cDNA. It can even amplify smaller mRNAs as well.
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 primer denaturation from the poly-A tail.
However, the oligo (dT) primers can not synthesize RNA other than mRNA because the tRNAs, rRNAs, and microRNA do not have the poly-A tail. 2 to 4 μM concentration of oligo (dT) primers are enough for RT-PCR, usually.
The binding of oligo (dT) primer on the template mRNA is shown in the figure below,
3. Sequence-specific primers:
The sequence-specific primers are commonly utilized in one-step RT-PCR to amplify a gene of interest. The sequences in the sequence-specific primers are complementary to the sequence of our interest therefore, they can’t amplify other gene regions.
Furthermore, it can only amplify a specific region. It needs a large number of primary templates to perform RT-PCR using this type of primers. It has direct utility in gene expression studies as it has higher sequence specificity.
See the figure below,
The sequence-specific primers synthesize only certain regions from the RNA, therefore, scientists recommend using less in order to achieve success in the reaction. 0.5 to 1.5 μM concentration is enough for the RT-qPCR.
Note: If you use sequence-specific primers and oligo (dT) primers both in a single reaction, use only 1μM each primer.
Reaction preparation for reverse transcription PCR:
Selecting every PCR ingredient and its quantity is as important as selecting temperature conditions for PCR. Nowadays, ready to use reverse transcription PCR kits make your work efficient as it has every ingredient in it. Let us see some components of RT-PCR,
The major components are,
- DNA primers
Read more on PCR primers: PCR primer design guidelines
- dNTPs
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
- Template nucleic acid
Cycling conditions for Reverse transcription PCR
We do not need the denaturation step here. The PCR reaction starts with the primer annealing. At the first stage, the primer binds to the template RNA, once it’s done, the reaction is placed for cooling at 4°C for proper binding. New strand synthesis initiates in stage two or the second step, following enzyme deactivation in step three.
Note: The length of primers, the composition of primers, the types of enzyme used in the reaction, and the length of the amplicon decide reaction temperature in each step.
The different steps of RT-PCR are shown in the figure:
Strand synthesis:
First-strand and second-strand cDNA synthesis:
The synthesis of cDNA is a crucial step in the entire RT-PCR protocol. The cDNA synthesis is completed into two steps, first-strand cDNA synthesis followed by second-strand DNA synthesis.
In the beginning, the reverse transcriptase enzyme amplifies the cDNA from the single-stranded RNA, known as a 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. Use E.coli RNase H separately into the reaction to separate the hybrid. Remember, do not use a higher concentration of RNase H because it cleaves the longer RNA template prematurely.
Now, the DNA polymerase fills the nicks generated by RNase H activity. Special types of enzymes such as DNA polymerase I and T7 govern the process of nick filling. The same DNA polymerase synthesizes DNA from the 5′ to 3′ direction by removing the RNA fragments in 5′ to 3′ exonuclease activity.
Enzyme ligase ligates nicks between adjacent DNA fragments after synthesis. Here, use E.coli DNA ligase instead of T4 DNA ligase. The second strand is now ready.
The figure below explains the whole process,
Note: The wild types of Moloney murine leukemia virus reverse transcriptase and Avian myeloblastosis virus reverse transcriptase is a good choice for RT-qPCR because both have adequate RNase H activity.
Applications:
- Reverse transcriptase PCR determines tissue-specific gene expression.
- The present method has been utilized to monitor the prognosis and therapy responses of cancer patients.
- It determines the amount of tissue-specific mutant alleles.
- It has important applications in gene insertion studies and gene therapy experiments.
- It also makes disease diagnosis and microbial identification possible by its novel approach.
- RT-PCR can measure viral load, expression, and infection.
Advantages of reverse transcription PCR:
- The method can do quantitative as well as qualitative analysis.
- It required a smaller amount of samples for gene expression studies.
- It doesn’t rely on post-PCR processing such as agarose gel electrophoresis.
- The method is simple, easy to use, rapid and cost-effective.
- Also, the specificity and the sensitivity of the reverse transcription PCR are enormous.
Disadvantages:
- The method is extremely sensitive, even a small amount of DNA contamination can lead to false results.
- The technique is used for some assays only.
- Huge experience and expertise are required to perform and develop new assays.
Read more,
- Multiplex PCR- Principle, Process, Protocol, Advantages, Limitations and Applications
- Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications
Reverse transcription data, results and interpretation:
We have explained one point somewhere in the above section, the assay doesn’t rely on post-PCR processing such as gel electrophoresis. Meaning the machine cat evaluates and executes results for us.
Understanding RT-PCR data needs a bit of experience. Here we obtain results in the form of a graph and data. The graph shows the number of amplification, cycles during amplification, the number of cycles amplified, etc.
The graph also shows data regarding the background score, unamplified target and other activities that happen during. Moreover, it can also show how many templates are correctly amplified.
For example, the Ct value or threshold cycle value shows in which cycles the amplification initiates, meaning, the amount of viral load present in a sample is inversely proportional to a number of cycles.
The data and results show,
- The amount of template
- The quality of the template
- Number of genes present (gene expression)
- Viral load in the sample
- Quantity of an allele
- Number of mutant and normal alleles
RT-PCR test for COVID-19:
One of my friends who is now in the COVID-19 testing told me that reverse transcription PCR has saved thousands or even more lives during the Pandemic. It’s a truly revolutionary diagnostic method.
The technique can measure the amount of viral load present in a sample by reverse transcription and amplification. In the technique, the performer first reverse transcribed the RNA into DNA.
Prepares the PCR reaction using a ready-to-use COVID-19 testing kit which usually has all the ingredients needed for RT-PCR.
Cycling conditions are set for 38 to 42 amplification cycles. The ct value of the COVID-19 RT PCR tells us the amount of viral load. Higher the load, lower the ct value and vice versa.
This is just an overview, you can read more on the whole process in this article:
Conclusion:
The reverse transcription PCR or RT-qPCR or qRT- PCR is a gold standard method for HIV and HPV detection. It can also detect other viral infections too.
It has unmatched applications in gene expression studies, transformation experiments and gene therapy and is also used to validate the ‘insert’ too.
The technique has nowadays been used often regularly in diagnostic and research laboratories across the world. In addition, the availability of various ready-to-use kits makes things easy for researchers. Nonetheless, experience and expertise are required to perform reverse transcription PCR and relevant assays.
Sources:
Mo Y, Wan R, Zhang Q. Application of reverse transcription-PCR and real-time PCR in nanotoxicity research. Methods Mol Biol. 2012;926:99-112. doi:10.1007/978-1-62703-002-1_7
Emery SL, Erdman DD, Bowen MD, et al. Real-time reverse transcription-polymerase chain reaction assay for SARS-associated coronavirus. Emerg Infect Dis. 2004;10(2):311-316. doi:10.3201/eid1002.030759
Hi sir… If there is acertification courses of pcr techniques?