RT-PCR: Definition, Principle, Enzymes, Types, Steps, Uses

Polymerase chain reaction (PCR) is a technique that allows scientists to make many copies of a specific DNA segment from a small amount of DNA. This technique is useful for studying DNA sequences, identifying genetic variations, diagnosing diseases, and detecting pathogens. PCR works by using a DNA polymerase enzyme that can synthesize new DNA strands from existing ones, following the rules of base pairing. However, the DNA polymerase enzyme needs a primer, a short piece of DNA that matches the beginning of the target DNA segment, to start the synthesis. Therefore, PCR requires two primers that flank the target DNA segment on both strands. By repeatedly heating and cooling the mixture of DNA, primers, and DNA polymerase, PCR can produce millions to billions of copies of the target DNA segment in a few hours.

Reverse transcriptase polymerase chain reaction (RT-PCR) is a variation of PCR that can amplify RNA instead of DNA. RNA is a molecule that carries genetic information from DNA to proteins in many organisms. However, RNA is less stable and more prone to degradation than DNA, so it is often difficult to study RNA directly. RT-PCR solves this problem by converting RNA into complementary DNA (cDNA) using an enzyme called reverse transcriptase. Reverse transcriptase can make a DNA strand that is complementary to an RNA strand, following the rules of base pairing. The cDNA can then be used as a template for PCR amplification, using the same principles as described above. RT-PCR is useful for studying gene expression, identifying RNA viruses, and detecting mutations in RNA.

In this article, we will explain the objectives, principle, enzymes, types, steps, uses, advantages, and limitations of RT-PCR in detail. We will also compare RT-PCR with other methods of RNA analysis and provide some examples of RT-PCR applications in different fields of biology and medicine.

RT-PCR is a powerful technique that combines the reverse transcription of RNA into complementary DNA (cDNA) with the amplification of specific cDNA targets using polymerase chain reaction (PCR). RT-PCR has several objectives, depending on the type and purpose of the analysis. Some of the main objectives are:

• To amplify and detect specific segments of RNA, such as mRNA, rRNA, viral RNA, or microRNA, resulting in billions of copies of a single RNA segment. This allows for the identification and quantification of RNA molecules in a sample, as well as the comparison of gene expression levels among different samples or conditions .
• To diagnose certain infections, genes, and diseases that are associated with RNA molecules. For example, RT-PCR can be used to detect and monitor RNA viruses such as HIV, SARS-CoV-2, hepatitis C virus, dengue virus, etc., as well as human genes involved in cancers, genetic disorders, or drug resistance .
• To study gene expression and regulation in various biological processes, such as development, differentiation, stress response, signaling pathways, etc. RT-PCR can measure the changes in mRNA levels in response to different stimuli or treatments, as well as the expression of alternative splicing variants, non-coding RNAs, or post-transcriptional modifications .
• To prepare cDNA libraries from eukaryotic mRNA samples for further analysis or manipulation. RT-PCR can generate cDNA molecules that lack introns and can be inserted into prokaryotic vectors for cloning, sequencing, or gene transfer .

RT-PCR is a versatile and widely used technique that has many applications in basic and applied research, clinical diagnosis and prognosis, and biotechnology. RT-PCR can provide accurate and reliable results with high sensitivity and specificity, as well as rapid and cost-effective performance .

RT-PCR combines the reverse transcription process with the conventional PCR process. The sample RNA is first converted to double-stranded DNA (complementary DNA) by reverse transcriptase enzyme in the reverse transcription process. The cDNA can then be thermally broken down into two single-stranded DNA templates. In these ssDNA templates, primers can anneal to their complementary sequences based on the nucleic acid hybridization principle. DNA polymerase then elongates the primer by sequentially adding the nucleotides to the 3’ end and generates a dsDNA following the principle of DNA replication. These three processes, denaturation, annealing, and elongation, are repeated in a cyclic manner regulating the reaction temperature and resulting in millions of copies of the cDNA.

The reverse transcription process involves the following steps:

• The sample RNA is mixed with a primer (oligo(dT), random or sequence-specific) and heated to 65°C for 5 minutes to denature any secondary structures.
• The mixture is cooled to 37°C and reverse transcriptase enzyme, dNTPs, RNase inhibitor and buffer are added. The reverse transcriptase enzyme synthesizes a complementary DNA strand from the RNA template using the primer and dNTPs as substrates. The RNase inhibitor prevents the degradation of RNA by RNases.
• The mixture is heated to 95°C for 15 minutes to inactivate the reverse transcriptase enzyme and degrade the RNA strand, leaving only the cDNA strand.

The PCR process involves the following steps:

• The cDNA is mixed with a pair of primers (forward and reverse), Taq DNA polymerase, dNTPs and buffer. The mixture is placed in a thermocycler that controls the temperature cycles.
• The first cycle starts with denaturation at 94°C for 30 seconds, where the cDNA is separated into two single strands.
• The second cycle starts with annealing at 50-60°C for 30 seconds, where the primers bind to their specific sequences on the cDNA strands.
• The third cycle starts with elongation at 72°C for 1 minute, where Taq DNA polymerase extends the primers by adding dNTPs to the 3’ end of each strand, resulting in two copies of dsDNA.
• The cycles are repeated for 25-40 times, doubling the number of dsDNA copies each time.

The principle of RT-PCR allows us to amplify a specific segment of RNA and detect its presence and quantity in a sample. It can be used for various applications such as gene expression analysis, viral detection, genetic diagnosis and gene therapy monitoring.

RT-PCR requires several enzymes and other components to carry out the reverse transcription and amplification processes. The main enzymes involved are:

• Reverse transcriptase enzyme: It is an enzyme that catalyzes the formation of complementary DNA (cDNA) strands from the RNA strand. It is also called RNA-dependent DNA polymerase enzyme and is responsible for reversing the central dogma of molecular biology. It is the major component of RT-PCR as it converts sample RNA into cDNA for amplification. There are different types of reverse transcriptase enzymes available, such as M-MLV RT, AMV RT, and SuperScript III RT, which vary in their temperature optimum, processivity, fidelity, and RNase H activity.

• RNase H enzyme: It is an enzyme that degrades the RNA strand in a RNA-DNA hybrid molecule. It is often present as a domain of reverse transcriptase enzyme or added separately in the reaction mixture. It is important for removing the RNA template after the synthesis of cDNA and preventing the formation of secondary structures that can interfere with the amplification process.

• DNA polymerase enzyme: DNA polymerases are enzymes that catalyze the synthesis of complementary DNA strands by assembling the nucleotides sequentially according to the template strand. Taq DNA polymerase, the DNA polymerase enzyme extracted from the bacterium Thermus aquaticus, is the most widely used DNA polymerase as it is thermally stable and continues its activity after the repeated cycle of heating and cooling. Other types of DNA polymerases, such as Pfu DNA polymerase and Vent DNA polymerase, have higher fidelity and proofreading ability than Taq DNA polymerase.

Besides these enzymes, RT-PCR also requires other components such as:

• Nucleic acid sample (sample RNA): RNA is the sample for RT-PCR, unlike other PCR techniques using DNA as their sample. Mostly mRNA is used as the sample. The RNA will be converted into cDNA before amplification. The quality and quantity of the sample RNA can affect the efficiency and accuracy of RT-PCR.

• Primers (oligo (dT) primers, random primers, and sequence-specific primers): Primers are short single-stranded sequences of nucleotides that bind to the complementary regions of the sample RNA or cDNA and provide a starting point for reverse transcriptase or DNA polymerase to extend. Three different types of primers are used in RT-PCR;

  • Random primers: These are short single-stranded sequences of 6 to 8 nucleotides that bind at random sites of RNAs with or without poly(A) tails for cDNA synthesis using reverse transcriptase. They can generate cDNA from all types of RNAs but may introduce non-specific amplification and primer-dimer formation.

  • Oligo (dT) primers: They are oligonucleotides, mostly of 12 – 18 nucleotides, containing a segment of repeating deoxythymidine (dT) which bind at the polyA tails of mRNA. They can generate cDNA from mRNA only but may miss some sequences at the 5` end of mRNA due to incomplete reverse transcription.

  • Sequence-specific primers: These are short single-stranded sequences of nucleotides that bind to the specific regions of interest of the sample RNA or cDNA. They can generate cDNA from specific RNAs only but may have higher specificity and sensitivity than random or oligo (dT) primers.

• Deoxynucleotide triphosphates (dNTPs): Deoxynucleotide triphosphates (dNTPs) are artificially synthesized nucleotides that act as building blocks for synthesizing cDNA and new cDNA strands during amplification. Four different dNTPs are used; deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), and deoxycytidine triphosphate (dCTP).

• PCR buffers and other chemicals: PCR buffers are solutions that provide optimal pH, salt concentration, magnesium ions, and other cofactors for the activity and stability of reverse transcriptase and DNA polymerase enzymes. Other chemicals such as DTT, EDTA, BSA, glycerol, etc., may also be added to enhance or inhibit certain reactions or prevent degradation or contamination.

• Thermocycler (PCR machine): A thermocycler is a device that can regulate the temperature of the reaction mixture according to a pre-programmed cycle. It is essential for carrying out the denaturation, annealing, and extension steps of RT-PCR in a precise and controlled manner.

Based on whether the reverse transcription and the amplification steps occur either in a single reaction (or tube) or in two separate reactions (or tubes), RT- PCR can be classified into two types: one-step and two-step.

One-Step RT-PCR

It is a type of RT – PCR where the reverse transcription and the amplification reactions occur in a single tube. All the required components are added in a single tube. First, reverse transcription occurs, forming cDNA, which is then amplified in a PCR process.

Advantages of One–Step RT – PCR over Two–Step RT – PCR

• It has a simple and easy handling setup.
• It has higher accuracy and specificity.
• It has a lesser chance of contamination.
• It is a cheaper and faster method.
• It is best suited for applications requiring high-speed and high-throughput amplification of many samples with few targets.
• Multiplex PCR of gene of interest and control can be done in single well, from same RNA sample.

Disadvantages of One-Step RT-PCR over Two-Step RT-PCR

• It detects fewer templates per reaction mixture due to using multiple chemicals in a single reaction tube.
• Due to lower template detection, it requires a larger template for starting.
• It does not permit the storage and further analysis of the cDNA formed during the reaction.
• There is a higher chance of primer – dimer and non–specific binding.
• The chance of reaction failure is comparatively high.
• The reaction conditions needed to support both the RT and PCR may not be optimal for either reaction, affecting efficiency and yield.

Two-Step RT-PCR

It is another type of RT – PCR where the reverse transcription and the amplification process occur in two separate tubes. In the first tube, a reverse transcription reaction takes place, yielding cDNA. These cDNAs are then transferred to another tube where the PCR mixture is added, and the cDNAs are amplified.

Advantages of Two-Step RT – PCR over One-Step RT – PCR

• It allows us to store cDNA formed by reverse transcription.
• It has higher efficiency, accuracy, and reliability and detects larger templates per reaction mixture.
• Greater flexibility to select RT enzymes and DNA polymerases for PCR separately.
• Store cDNA for later use.
• Preferred method for applications with limited amount of starting material (i.e. single cell analysis).
• It provides greater sensitivity and flexibility, but reduces throughput.

Disadvantages of Two–Step RT – PCR over One-Step RT – PCR

• There is a higher chance of contamination.
• It is a more complex and tedious process requiring more resources and a well-trained person to operate.

One-step and two-step RT-PCR are two different types of RT-PCR based on whether the reverse transcription and the amplification steps occur in a single tube or in two separate tubes. Both methods have their own advantages and disadvantages depending on the purpose and the conditions of the experiment.

One-Step RT-PCR

One-step RT-PCR is a simpler and faster method than two-step RT-PCR as it requires only one reaction tube and one set of primers. It also reduces the risk of contamination and sample loss as there is no need to transfer the cDNA to another tube. One-step RT-PCR is more suitable for high-throughput screening, multiplexing, and real-time detection of RNA targets.

However, one-step RT-PCR also has some drawbacks. It may have lower sensitivity and specificity than two-step RT-PCR as it uses multiple enzymes and chemicals in a single reaction tube, which may interfere with each other or degrade over time. It also requires more optimization of the reaction conditions and the primer design to avoid non-specific amplification or primer-dimer formation. Moreover, one-step RT-PCR does not allow the storage and further analysis of the cDNA, which may limit its applications in some cases.

Two-Step RT-PCR

Two-step RT-PCR is a more sensitive and reliable method than one-step RT-PCR as it uses separate tubes and primers for the reverse transcription and the amplification steps. It also allows more flexibility in choosing different types of primers, enzymes, and buffers for each step to optimize the reaction efficiency and specificity. Two-step RT-PCR is more suitable for low-abundance or rare RNA targets, nested PCR, and cloning of cDNA.

However, two-step RT-PCR also has some disadvantages. It is more complex and time-consuming than one-step RT-PCR as it requires two reaction tubes and two sets of primers. It also increases the chance of contamination and sample loss as it involves transferring the cDNA to another tube. Moreover, two-step RT-PCR requires more resources and skills to perform and analyze the results.

Therefore, one should consider the advantages and disadvantages of both methods before choosing the most appropriate one for their experiment. Some factors that may influence the decision are:

• The quantity and quality of the RNA sample
• The type and size of the RNA target
• The purpose and scope of the experiment
• The availability and cost of the reagents and equipment
• The level of expertise and experience of the experimenter
  
  

  Steps/Procedure of RT-PCR

The core procedure of RT-PCR can be broadly classified into two phases: reverse transcription and amplification. The procedure also varies depending on whether one-step or two-step RT-PCR is used. But, the general steps involved in both types are the same and can be summarized into four stages: preparatory stage, reverse transcription, amplification, and product analysis stage.

• Preparatory stage: It is the initial stage where RNA extraction is done, and all the reaction mixture is prepared. First, all materials are arranged, safety measures are taken, the PCR reaction preparation area is cleaned, all the reagents are brought to working temperature, and the sample is extracted or brought from storage.

  • In one-step RT-PCR, sample RNA, reverse transcriptase enzyme, RNase H, primers, DNA polymerase, dNTPs, buffers, and all other components are added in a specified and pre-calculated amount in a single reaction tube. The tube is then loaded into a thermocycler for further processing.

  • In two-step RT-PCR, sample RNA, reverse transcriptase enzyme, RNase H, primers, dNTPs, and other buffers and chemicals for reverse transcription are loaded in a tube. Then the tube is subjected to a specified temperature in a thermocycler where cDNAs are formed.

• Reverse transcription: It is the primary step where the RNA is converted into cDNA using a reverse transcriptase enzyme.

  • All the reaction mixture, including reverse transcriptase enzyme, RNase H, dNTPs mixture, primers, nuclease-free water, reverse transcription buffer, and other components in one-step RT-PCR and DNA polymerase and other amplification components in two-step RT-PCR are added in a tube and subjected to a temperature of 40–50°C for 10 minutes to 30 minutes in a thermocycler. At this temperature, the primer will bind to the respective site of the RNA sample, and the reverse transcriptase enzyme will synthesize cDNA by adding the free dNTPs .

• Amplification: This step is similar to the amplification process of other PCR techniques for DNA amplification.

  • In one-step RT-PCR, the same reaction mixture is subjected to an amplification process. At the same time, in two-step RT-PCR, the cDNA is isolated and placed in another tube where DNA polymerase enzyme, primers, PCR buffer, dNTPs, and other chemicals are added. Then the tube is placed in a thermocycler for amplification.

  • The amplification step includes denaturation (heating to 94°C for 15–30 seconds), annealing (lowering to 54–60°C for 20–40 seconds), and elongation/extension (raising to 72–80°C for 30–60 seconds) occurring cyclically one after another for a certain number of cycles pre-programmed by the user .

• Product analysis stage: It is the final step where the reaction mixture subjected to PCR is analyzed to confirm that desired amplification is achieved. The gel electrophoresis method is mostly used for product analysis. In real-time RT-PCR (qRT-PCR), there is no need for this additional step as fluorescence is used to monitor the amplification reaction .

Applications of RT-PCR

RT-PCR is a versatile technique that has many applications in research and clinical settings. Some of the most common applications are:

• Study gene expression: RT-PCR can measure the amount and quality of mRNA transcripts in a cell or tissue, which reflects the level of gene expression. RT-PCR can also detect alternative splicing events, gene fusions, and mutations that affect mRNA structure and function. RT-PCR is widely used to study the regulation and function of genes in various biological processes, such as development, differentiation, stress response, and disease.

• Identification of unknown species: RT-PCR can identify and classify microorganisms and other organisms based on their rRNA and mRNA sequences. RT-PCR can also detect novel or emerging pathogens that are not easily cultured or characterized by conventional methods. RT-PCR has been used to identify viruses such as HIV, SARS-CoV, dengue virus, Hantavirus, human metapneumovirus, and West Nile virus.

• Infectious disease diagnosis: RT-PCR can diagnose various types of viral, bacterial, fungal, and parasitic infections by detecting the presence and quantity of specific pathogen RNA in clinical samples. RT-PCR can also monitor the response to treatment and the emergence of drug resistance by measuring changes in viral load or gene expression. RT-PCR is a rapid, sensitive, and specific method for the detection of RNA viruses and infections by them.

• Gene insertion and gene therapy study: RT-PCR can prepare cDNA from eukaryotic mRNA, which lacks introns and can be inserted into prokaryotes for genetic engineering purposes. RT-PCR can also monitor the result of gene insertion and gene therapy by analyzing the expression and function of the inserted or modified genes. RT-PCR can detect tissue-specific mutant alleles and unique mRNAs produced by different types of cancer cells or gene therapy recipients.

• Tools of genetic engineering and viral study: RT-PCR can amplify target RNA for further analysis by cloning, sequencing, microarray, or RNA-seq. RT-PCR can also generate cDNA libraries from various sources of RNA for screening and discovery purposes. RT-PCR can study the structure, function, evolution, and interaction of viral genomes and transcripts by comparing different strains, isolates, or mutants.

Advantages and Limitations of RT-PCR

RT-PCR is a powerful technique that combines the reverse transcription of RNA into cDNA with the amplification of the target cDNA by PCR. It has many applications in research, diagnosis, and biotechnology. However, it also has some limitations and challenges that need to be considered. Here are some of the advantages and limitations of RT-PCR:

Advantages of RT-PCR

• It is a very rapid method for amplifying RNA and can enzymatically produce millions of copies of mRNA in a very short time.
• It is very simple to operate. The process is semi-automatic, operated, and regulated by a thermocycler without human involvement.
• It has very high specificity and sensitivity but is economical. It can detect and quantify very low levels of RNA in a sample, even in the presence of other nucleic acids or contaminants.
• It is a very accurate method for the identification of RNA viruses and infection by them. RNA viruses can be classified up to the level of strains. It has shortened the time for identifying RNA viruses and viral infections.
• It can detect a very minute amount of mRNA (about 5pg) compared to the traditional Northern Blot technique. It can also analyze the expression of multiple genes simultaneously using multiplex RT-PCR or real-time RT-PCR.
• Mutated genes and gene expression can be easily and promptly studied. This has made it possible to diagnose cancer in the early stage, study gene insertion, and monitor the result of gene therapy.
• It is both a qualitative and quantitative method; hence can be used to identify as well as quantify the sample RNA. Real-time RT-PCR can provide real-time data on the amplification process and calculate the relative or absolute expression levels of the target gene.

Limitations of RT-PCR

• It can amplify RNAs only, especially mRNAs. Other types of RNAs, such as rRNAs or tRNAs, may interfere with the reverse transcription or amplification process. Therefore, RNA purification and quality control are essential steps before performing RT-PCR.
• Prior information regarding the sequence of the RNA is required for primer designing. The primers must be specific and complementary to the target RNA sequence. Otherwise, they may anneal to non-target regions or form primer-dimers, leading to false results or reduced efficiency.
• It is a full temperature and enzyme-based system, so a slight change in the reaction temperature will decrease the efficiency of the enzyme. Hence, it requires a strict temperature regulation system and optimal reaction conditions for each step.
• Slight contamination, having a similar primer binding site, can be amplified, giving a false positive or false negative result. Therefore, proper precautions must be taken to avoid cross-contamination or carry-over contamination between samples or reagents.
• The reaction can be highly influenced by a minute amount of organic or inorganic contaminant in the reaction mixture. For example, inhibitors such as heparin, hemoglobin, phenol, ethanol, or EDTA can affect the activity of reverse transcriptase or DNA polymerase enzymes. Therefore, careful extraction and purification of RNA are necessary to remove any potential inhibitors.
• The process is very tedious, requiring a complex reaction mixture and a skilled person to operate. It also requires specialized equipment and reagents that may not be easily available or affordable in some settings.

RT-PCR is a versatile technique that has many applications in research and clinical settings. Some of the most common applications are:

• Study gene expression: RT-PCR can measure the amount and quality of mRNA transcripts in a cell or tissue, which reflects the level of gene expression. RT-PCR can also detect alternative splicing events, gene fusions, and mutations that affect mRNA structure and function. RT-PCR is widely used to study the regulation and function of genes in various biological processes, such as development, differentiation, stress response, and disease.

• Identification of unknown species: RT-PCR can identify and classify microorganisms and other organisms based on their rRNA and mRNA sequences. RT-PCR can also detect novel or emerging pathogens that are not easily cultured or characterized by conventional methods. RT-PCR has been used to identify viruses such as HIV, SARS-CoV, dengue virus, Hantavirus, human metapneumovirus, and West Nile virus.

• Infectious disease diagnosis: RT-PCR can diagnose various types of viral, bacterial, fungal, and parasitic infections by detecting the presence and quantity of specific pathogen RNA in clinical samples. RT-PCR can also monitor the response to treatment and the emergence of drug resistance by measuring changes in viral load or gene expression. RT-PCR is a rapid, sensitive, and specific method for the detection of RNA viruses and infections by them.

• Gene insertion and gene therapy study: RT-PCR can prepare cDNA from eukaryotic mRNA, which lacks introns and can be inserted into prokaryotes for genetic engineering purposes. RT-PCR can also monitor the result of gene insertion and gene therapy by analyzing the expression and function of the inserted or modified genes. RT-PCR can detect tissue-specific mutant alleles and unique mRNAs produced by different types of cancer cells or gene therapy recipients.

• Tools of genetic engineering and viral study: RT-PCR can amplify target RNA for further analysis by cloning, sequencing, microarray, or RNA-seq. RT-PCR can also generate cDNA libraries from various sources of RNA for screening and discovery purposes. RT-PCR can study the structure, function, evolution, and interaction of viral genomes and transcripts by comparing different strains, isolates, or mutants.

RT-PCR is a powerful technique that combines the reverse transcription of RNA into cDNA with the amplification of the target cDNA by PCR. It has many applications in research, diagnosis, and biotechnology. However, it also has some limitations and challenges that need to be considered. Here are some of the advantages and limitations of RT-PCR:

Advantages of RT-PCR

• It is a very rapid method for amplifying RNA and can enzymatically produce millions of copies of mRNA in a very short time.
• It is very simple to operate. The process is semi-automatic, operated, and regulated by a thermocycler without human involvement.
• It has very high specificity and sensitivity but is economical. It can detect and quantify very low levels of RNA in a sample, even in the presence of other nucleic acids or contaminants.
• It is a very accurate method for the identification of RNA viruses and infection by them. RNA viruses can be classified up to the level of strains. It has shortened the time for identifying RNA viruses and viral infections.
• It can detect a very minute amount of mRNA (about 5pg) compared to the traditional Northern Blot technique. It can also analyze the expression of multiple genes simultaneously using multiplex RT-PCR or real-time RT-PCR.
• Mutated genes and gene expression can be easily and promptly studied. This has made it possible to diagnose cancer in the early stage, study gene insertion, and monitor the result of gene therapy.
• It is both a qualitative and quantitative method; hence can be used to identify as well as quantify the sample RNA. Real-time RT-PCR can provide real-time data on the amplification process and calculate the relative or absolute expression levels of the target gene.

Limitations of RT-PCR

• It can amplify RNAs only, especially mRNAs. Other types of RNAs, such as rRNAs or tRNAs, may interfere with the reverse transcription or amplification process. Therefore, RNA purification and quality control are essential steps before performing RT-PCR.
• Prior information regarding the sequence of the RNA is required for primer designing. The primers must be specific and complementary to the target RNA sequence. Otherwise, they may anneal to non-target regions or form primer-dimers, leading to false results or reduced efficiency.
• It is a full temperature and enzyme-based system, so a slight change in the reaction temperature will decrease the efficiency of the enzyme. Hence, it requires a strict temperature regulation system and optimal reaction conditions for each step.
• Slight contamination, having a similar primer binding site, can be amplified, giving a false positive or false negative result. Therefore, proper precautions must be taken to avoid cross-contamination or carry-over contamination between samples or reagents.
• The reaction can be highly influenced by a minute amount of organic or inorganic contaminant in the reaction mixture. For example, inhibitors such as heparin, hemoglobin, phenol, ethanol, or EDTA can affect the activity of reverse transcriptase or DNA polymerase enzymes. Therefore, careful extraction and purification of RNA are necessary to remove any potential inhibitors.
• The process is very tedious, requiring a complex reaction mixture and a skilled person to operate. It also requires specialized equipment and reagents that may not be easily available or affordable in some settings.

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