Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications

“A technique used to quantify the nucleic acid (DNA/RNA) present in a sample, during the PCR reaction is known as a real-time PCR or quantitative (q)PCR”

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In other words, we can say,

“A molecular biology technique used to monitor the amplification of the target DNA/RNA sequence is referred to as real-time PCR or quantitative PCR.”   

We know many things about PCR but distinguishing RT-PCR from conventional PCR is quite a difficult task for some newbies. We have many articles on the present topic, ranging from explanations on PCR to how to optimize the annealing temperature for PCR. You can read by searching a keyword in the search box.

Amplifying a template isn’t sometimes enough to conclude results, especially for microbial detection, infection study and gene expression studies.

RT-PCR or qPCR is a different concept that actually measures and/or quantifies each and every template present in the sample. In this context, I will explain each and every point related to the real-time PCR in this blog post. Common topics will be an introduction to the technique, Principle, procedure, advantages, disadvantages and applications.

I have tried to explain everything pictorially as well as theoretically. Besides, the present article consists of other related information too and so is huge! take a cup of tea and enjoy the article.

Stay tuned,

Abbreviations:

Abbreviation	Full name
PCR	Polymerase chain reaction
qPCR	Quantitative PCR
rt PCR	Real-time PCR
RT- PCR	Reverse transcriptase PCR
FRET	Fluorescent resonance energy transport
Taq Man	Taq DNA polymerase (Pac) Man

What is Real-Time PCR?

Amplification is the prime goal of any PCR reaction. Using dNTPs, primers and PCR reaction buffer, the Taq DNA polymerase amplifies our DNA in vitro. The same process when occurs in vivo, known as replication.

In the traditional PCR method after the amplification, the PCR products or the amplicon are run on the agarose gel or PAGE to detect the presence or absence of DNA amplification. But in the real-time PCR monitor amplification after each PCR cycle in a real-time manner.

A camera or detector detects each amplicon produced during the amplification by measuring the fluorescence emitted.

Usually, the chemistry behind real-time monitoring relies on the use of fluorescent dye. As the amplification progresses, the detector detects the amount of fluorescence emitted. This is the basic and global principle behind all types of real-time PCR.

However, some platforms use labeled probes instead of dNTPs.

Before understanding the principle more in-depth, let us first know why the real-time PCR is more advanced?

Why is real-time PCR more advanced than traditional conventional PCR?

The reason is simply clear, we can calculate or measure how much templated is amplified which a conventional PCR can’t do. And this is the reason the technique is also named “quantitative PCR.”

In the conventional PCR, we have to do agarose gel electrophoresis that is also not required here. By doing the melting curve analysis one can get results. Moreover, one can choose which reaction is performing well through running parallel reactions.

Only a well-performing reaction counts. This minimizes the chances of error. Now, immediately a question strikes in mind, how can it help?

The primer application of the technique is to analyze gene expression, which absolutely isn’t possible by conventional PCR. Tissue-specific gene expression; the number of every gene present can be monitored, everything happens in real-time.

Other applications will be covered in the upcoming section, but before that let us understand the principle.

The principle of real-time PCR:

The principle of real-time PCR relies on the use of fluorescent dye. In general, the principle of the present method is stated below,

“The amount of the nucleic acid present into the sample is quantified using the fluorescent dye or using the fluorescent-labeled oligos.” When a dye or probe binds with the target template, it releases a fluorochrome which resultantly emits fluorescence for the detector to detect. The detector captures a signal as a positive template amplification.

Although, it isn’t as simple. Let me explain things in-depth. Two types of chemistry are available for the real-time quantitative PCR:

1. DNA binding dye (Intercalating dye-based method)
2. Sequence-specific probe (Hydrolysis Probe-based detection method)

DNA binding dye:

For a novice and inexperienced person, the DNA binding dye method is the best technique for real-time detection.

The dye has its own fluorescence. Once the dye binds to the double-stranded DNA the fluorescence emitted by the dye increases 100 to 1000 fold than the original signal. However, the original dye fluorescence is taken as the baseline (as a reference) for the detection.

The method is rapid, quick, reliable and cost-effective. Also, the chance of error in the experiments is less and the reaction setup is simple & easy to use.

The result of the experiment depends on the specificity of the primers used in the PCR reaction. Because even though the primers remain bound non-specifically, the DNA binding dye binds to the non-specific sequence and gives the fluorescent signals.

As the dye detects the double-stranded DNA to bind, even if the dsDNA is non-specific, the dye binds to it. Therefore the chance of the non-specific detection is high in the SYBR green dye-based method. The SYBR green is one of the most popular dyes used in real-time PCR.

The sensitivity of the experiment is limited. Again a question arises in mind,

Is it suitable for the determination of sensitive templates?

The answer is Yes,

A melting curve analysis helps to identify non-specific bindings during the reaction.

Melting curve analysis:

After completion of the amplification reaction and capturing fluorescence signals, melting the template (again) determines non-specific bindings if any. During melting, at a high temperature, the template starts denaturing which consequence dye dissociation and reduce fluorescence.

Varied heat transition reported shows the amount of non-specific products while the gradual decrease in fluorescence shows the presence of specific amplification product.

Put simply, the story tells that,

“A larger sequence need more time and higher temperature for melting while non-specific amplicons needs lower and varied temperature to melt and so gives more shorter curves in a graph.

I have tried putting this explanation as a graph, you can see the fluorescence vs melting temperature graph below,

The fluorescence vs melting temperature graph is also called a dissociation curve and the method is called a dissociation curve analysis.

The first image shows the presence of primer-dimer while the second image shows the dissociation curve of different alleles at different temperatures.

Two common dyes employed in the real-time PCR are the SYBR green and EvaGreen, notedly, the technique is used in the validation of other assays such as DNA microarray.

Probe-based detection method:

The method used the single short sequence-specific probes which are of two types:

In the probe-based detection method, two different types of single and short-sequence-specific probes are utilized; A linear probe and molecular beacons.

Linear probe:

Linear probes are the TaqMan probe, which relies on the activity of Taq DNA polymerase. The probes structurally consist of labeled short single-stranded sequence-specific DNA molecules that are radio or fluorescent-labeled.

Here the probe is labeled with the fluorescent dye described as a reporter molecule, situated at the 3’ end. The other 5’ end has the quencher dye which is in close proximity to the reporter dye and quenches the fluorescence of the reporter dye.

Now, in the probe base method, not only the probe but the Taq DNA polymerase plays an important role. The Taq DNA polymerase used in the real-time PCR has the 5’ to 3’ exonuclease activity, which removes the probe by extending the DNA.

Once the probe dissociates the reporter molecules emitted fluorescent light. Because, if the DNA (the sequence of our interest) is amplified, the reporter molecule is unquenched and releases the fluorescence.

See the figure below,

The main advantage of the probe-based method is that we can use multiple probes for multiple template DNA sequences. This means we can amplify multiple templates in a single reaction efficiently.

TAMRA and Black Hole Quencher are two widely used quencher dyes. While FAM is the most popular reporter dye.

From a technical point of view, we have to face one problem, with this! which indeed limits the use of the probe-based technique. The same annealing temperature is not possible for both- primer as well as a probe.

It’s a big problem for many reactions. However, the use of lower extension temperature can help. So basically, using only two steps, instead of three, the problem can be solved.

What we do is the annealing and the extension step are combined at 60°C. After the denaturation, the probe hybridization, primer binds and extension is done at a single temperature.

 At 72°C extension, the Taq will be at its highest activity therefore, instead of removing the probe it facilitates strand displacement of a probe. That is why the annealing and the extension in the linear probe-based real-time PCR are done at a single temperature.

We will prepare a whole article on the TaqMan probe and oligo probes and discuss all the advantages and disadvantages there.

“After each PCR cycle, more probes are hydrolyzed, more fluorescence is generated and more amplicons are quantified.”

We had written two amazing articles on Taq DNA polymerase, read it here:

1. Choosing the right DNA polymerase for your PCR experiment
2. Function of Taq DNA polymerase in PCR

Molecular beacons:

The molecular beacons rely on the mechanism of thermodynamics in which a molecule remains in such a condition where the majority of its energy can be saved. Here instead of binding non-specifically, the molecular beacon remains in a hairpin structure.

What a molecular beacon facilitate is that preventing non-specific binding during the reaction which is commonly observed while using linear probes.

Structurally, the complementary sequences present on both ends of the hairpin loop-like structure helps to prevent non-specificity. on the other hand, the central loop is complementary to the target sequences. One end of the hairpin loop has the quencher dye and one end has the reporter fluorescent dye.

Here, interestingly, when the two ends of the hairpin stem are in close proximity with each other, the reporter molecule remains quenched and cannot generate fluorescence. But when it binds to the complementary sequence, the two ends of the hairpin separate from each other, the quencher blocks, the reported dye is released and emits the fluorescence.

The detector records emission, the image below explains show thing.

The molecular beacon probes are highly sequence-specific and are the best choice for sensitive reactions. If the probe (molecular beacon) cannot find its complementary sequence, it remains in the hairpin loop form and prevents non-specific bindings.

It can’t compromise the sensitivity of the PCR as the mechanism does not rely on the activity of Taq DNA polymerase. And so unlike the dissociation curve analysis (which completes in two steps), the whole reaction completes in three separate steps that facilitate Taq Pol to work at its full activity at 72°C.

In molecular beacon chemistry, the structure of the beacon stem is very important. Designing the loop for the beacon is a crucial step for getting specific amplification.

Suppose, if the structure of the hairpin loop is too stable, it can’t be separated, can’t unquench, can’t do hybridization and fails reaction. Melting curve analysis is necessary to assist the function of the molecular beacons.

We have covered a dedicated article on the molecular beacon, you can read it here: Molecular Beacon: A hairpin that enhances real-time PCR specificity.

Scorpion probes:

Scorpion probes are other types of probes or we say, a type of molecular beacon in which instead of two different probes and primer, the hairpin loop is incorporated directly at the 5′ end of the primer. The 3′ end contains the complementary sequence to our target DNA.

The scorpion probe is even more specific than the molecular beacons. but you may wonder How can we decide which method is used for which types of real-time experiments? Or can we use both methods for all the samples?

Well, that depends on,

1. What types of experiments do we want to perform.
2. The overall cost or the budget of our sample.
3. The knowledge and expertise of the researcher and
4. The tissue type of the sample.

Steps and procedure of real-time PCR:

The quantification is achieved by amplifying and monitoring the DNA or RNA present in the sample. For the quantification of the gene expression, the RNA is quantified into the real-time PCR.

If DNA is present in the sample in a higher quantity, amplification and quantification start at the early stage of the reaction; otherwise, the amplification starts in the late stage.

As like the conventional PCR, there are three main steps in real-time PCR;

1. Denaturation
2. Annealing
3. Extension

Denaturation occurs at 94°C where the double-stranded DNA is denatured and two single-stranded DNA is generated. The DNA is melted.

This single-stranded DNA is the sight of the annealing for the primers in the later step of the amplification.

Annealing occurs at 55°C to 66°C in which the sequence-specific primer bind to the single-stranded DNA. Along with it, the fluorescent dye or the probe bind to the DNA sequence too. 

Extension occurs at 72°C at which the Taq DNA polymerase activated highest. In this step, the Taq adds dNTPs to the growing DNA strand.

Note: if the amplicons are less, combine the extension step with the annealing step (for real-time PCR only).

Components used into the real-time PCR:

Similar to conventional PCR, the real-time PCR reaction contains almost the same components except for the fluorescent dye or fluorescent-labeled probe.

Let’s start with the dNTPs

dNTPs:

dNTPs are added during the synthesis of the growing DNA strand by the Taq DNA polymerase. The dNTPs remain the same as the conventional PCR.

For more detail read the article: The Function of dNTPs in PCR reaction

Taq DNA polymerase:

Normal Taq cannot work efficiently for the real-time PCR, instead always use the hot-start Taq DNA polymerase. The hot start Taq DNA polymerase is the best choice for the quantification.  

Read our article on Hotstart PCR: What is a hot-start PCR?

MgCl2:

Magnesium ion also plays a crucial role in the amplification during real-time PCR. However, the concentration of the Mg2+ ions is different from the conventional PCR. Use 3 to 5mM of MgCl2 in the real-time PCR.

Read the article on MgCl2: Role of MgCl2 in PCR reaction

Template:

100pg to 1microgram template DNA is sufficient for real-time PCR. We required only 100 copies of genomic DNA/RNA fragments for the amplification and to start the reaction. The template DNA or RNA must be pure and free from any contaminants. 

Read more on template DNA: What is the properties of PCR (template) DNA?

Primers:

Do not worry about the primers of real-time PCR. Use ready-to-use primer kits. If you want to design the primers by yourself keep several points in mind,

The primer should be shorter, it can amplify only 100 to 160bp fragments, and avoid longer amplicons.

It must be unique and contains 50% GC contains with the GC sequences at the end of the 3’ end.

It should be 18 to 20 nucleotides long.

The primer contains all these criteria that are the best for a real-time PCR assay.

For more detail read our primer design guidelines: PCR primer design guidelines.

Note: In each section, we had given the link of related articles for more detail. You can read it to understand the topic better.

The procedure of the real-time PCR starts with the extraction. The DNA or the RNA is extracted and quantified using the ready-to-use kits.

Now, prepare the reaction as per the manufacturer’s protocol, care must be taken while preparing the reaction.

Set the cyclic condition of the PCR and put the samples inside the machine. After the amplification, standard curve analysis or relative quantification is performed, instead of agarose gel electrophoresis.

For quantification of different types of samples, Endpoint PCR or real-time PCR can be performed.

Endpoint PCR vs Real-time PCR:

The main fundamental behind the real-time PCR is around the intensity of the fluorescence emitted whether it is emitted during the PCR or at the end of the PCR.

In the endpoint PCR, quantification is performed after the completion of the PCR reaction.

Based on the total fluorescence emitted, the amount of template is determined into the sample. The method is also called as semi-quantitative PCR.

However, this method has one major problem.

At the later stage of the amplification the reagents available for the amplification are less (because it is consumed during the early reaction) also the amplification inhibitors are active more. Hence accurate measurement is not possible in this method.

Therefore this method is inconsistent and inaccurate.

Even if we amplified the identical sample multiple times, the result does not remain the same in all reactions.

The end-point semi-quantitative method is best for just confirming the amplicons, it is not suitable for the gene expression and viral titer measurements. 

However, the method is rapid and faster than conventional PCR.

The real-time quantitative PCR is more sensitive and accurate than the endpoint PCR.

Because here, the amplification is measured in real-time, during the reaction. After each reaction, the fluorescence is emitted and it is reported by the detector.

The signals are recorded during the exponential phase of the reaction.

The figure below is the graph of a real-time PCR reaction,

Here, the amplification is not recorded during the late phase of the reaction. The reason is the same as the endpoint PCR.

The real-time PCR method is undoubtedly more accurate and reliable than other methods.

It is used for the quantification of DNA, RNA and gene expression.

The sample source for the real-time quantification is gDNA, cDNA, RNA, Gene of interest, synthetic oligos, total RNA or plasmid DNA.

The real-time or quantitative analysis is divided into two  other methods:

• Standard curve analysis
• Relative quantification

Standard curve analysis:

In the standard curve analysis method, the serially diluted sample or template is quantified against the known template.

Here the known template is serially diluted many times and quantified. The source of the information is used for the sample and unknown template which is also serially diluted and measured against the known.

In simple words, we can say that each unknown sample dilution is compared with each known standard dilution.

The method is also called absolute quantification. The method is one of the best choices for the viral load quantification and bacterial load quantification present into the sample. Also, the absolute quantification method is rapid and more accurate.

By comparing the Ct value of both the standard and the unknown template, the linear curve graph is generated.

For each know and unknown dilution, the Ct of all is plotted on the graph and by comparing the data the initial concentration of the unknown template is determined.

However, the method of calculation contains so much maths, hence we are not discussing it but it is automatically calculated by the machine.

Relative quantification:

Another method is for those types of the template which do not have reference value. Or it is totally unknown.

Here, the calibrator is used to create the baseline for the experiment, and with respect to the baseline calibrate and the Ct value of the template sample, the amount of the expression of the gene into the unknown sample can be determined.

The figure below shows the graph of different templates,

Common terminologies:

Ct value	The point at which the fluorescent is measurable.
Baseline	Point of the initial amplification where the fluorescent is nearly zero. No template zone.
Threshold	A threshold is a set of single which distinguish amplification signals from the background signals
Background signals	Signals of unamplified DNA
Exponential phase	The phase at which the reported amplification is at its highest peak.

Read further,

1. DNA sequencing
2. What is gene editing and CRISPR-CAS9?

Advantages of Real-time PCR:

The method is cost-effective.

The conventional PCR method is more costly than the qPCR due to the use of so many other chemicals and agarose gel electrophoresis.

It is time-efficient.

Definitely, it is.

The average time consumed by the PCR reaction along with the agarose gel electrophoresis and data interpretation is approximately 4 to 4.5 hours.

Contrary, real-time qPCR gives results in ultra-fast time. The average duration of the qPCR reaction is around 30 minutes to 2 hours.

More sensitivity and specificity.

The quantitative real-time PCR method is more sensitive, specific and efficient.

Though the probes and primers are highly sequence-specific, if any non-specific bindings occurred, it is monitored immediately during the reaction. Also, the main reaction or the quantification of our template cannot be influenced by the non-specific bindings.

Fewer templates required:

The overall assay required less amount of the template material. It required 1000 folds less template DNA or RNA for the reaction to occur as compared with the conventional PCR.

Melting curve analysis:

The main advantage of quantitative PCR is the confirmation of the analytes through the melting curve analysis.

We can measure and quantify how many amplicons are generated and how many non-specific or primer-dimers are formed during the PCR reaction by doing the melting curve analysis.  

The major advantage over the other PCR technique is the quantification. It quantifies the template DNA or RNA present in the sample.

Only Real-time is sufficient:

No post PCR processing and data processing is required in the quantitative real-time PCR. As with conventional PCR, agarose gel electrophoresis gel-based interpretation is not needed in the qPCR.

Limitation of real-time qPCR

Although the advantages of the quantitative rt PCR are far more than the conventional PCR, still the technology has several limitations.

The instrument itself is too costly as compared with conventional PCR.

Also, the multiplexing is still limited in Real-time PCR.

Kits are not available for all kinds of genes and disorders. The technical and standardized protocols are limited. Furthermore, higher expertise and technical skills are required for developing a novel qPCR assay.

Applications of quantitative PCR:

From gene quantification to gene expression, the real-time qPCR is the ocean of different applications in different fields. Apart from gene expression studies, it is further used in food industries, microbial identification and disease diagnosis.

Disease diagnosis:

One of the most powerful uses of any PCR technique is in the inherited disease diagnosis.

The real-time PCR is used in the diagnosis of a single gene and multigenic disease. It is used to quantify the mutated gene in the disease patient. The quantitative real-time PCR is even used in the determination of copy number variation in different tissues for different inherited disorders.

microRNA analysis:

MicroRNAs are smaller RNA molecules of 20 to 25 nucleotides in length. It plays an important role in the gene regulation pathway. The qPCR assay is used to quantify the microRNA from different tissues.

By quantifying it we can estimate the gene expression level in different tissues influenced by the microRNA.

Cancer detection:

Circulating tumor cells contain the mutant mRNA which transports to different tissues if it is malignant.

By quantifying the total mRNA against the mutant mRNA into the samples of cancer or from the cancer cell. The stage of cancer can be determined. By which the severity of the carcinoma can be estimated.

The mutant mRNA is the best biomarker for gene expression studies for cancer.

Furthermore, real-time quantification can be helpful in measuring the recovery in cancer therapy. After each therapy, the gene expression level of the mutant oncogenic cancer genes from the affected tissues is determined. 

By doing rt PCR, the success of the therapy can be estimated.

Therefore, cancer diagnosis, prognosis and monitoring to the response of therapy can be done using the real-time qPCR.

Microbial load testing:

The accurate microbial load testing from any biological sample is nearly impossible without the qPCR.

Microbial load in the fermented sample, soil sample, water sample, food and food spoilage can be accurately estimated by the real-time PCR.

Also, the estimation of the active microbial load is determined. Additionally, microbial risk assessment can also be possible by this method.

GMOs detection:

Genetically modified organisms are the organism whose genetic makeup is altered using the genetic engineering or transgenic technique specifically into plant, animal and microorganism.  

Now, this is something interesting,

Inserting DNA through the vector is not sufficient to do so. For the successful development of GMOs, one has to estimate the expression or the protein formed by the inserted gene.

For that, only gene detection cannot help, the mRNA expression quantification through real-time PCR helps to detect the amount of the gene expressed into the GMO.

Further, the amount of inheritance of that inserted gene is determined as well.

Genotyping and quantification of pathogens:

Microbial infections are the second most common reason for worldwide mortality and morbidity. Unlike conventional PCR (which can only detect a single or a few strains and genotyping), RT-PCR can quantify the amount of infection and measure microbes present in a sample.

Melting curve analysis, dissociation curve analysis and Ct value analysis help in investigating the severity of the infection. The most recent examples are the detection and quantification of COVID-19 coronavirus and H1N1 swine flu.

q(RT)- PCR helped in both types of pandemics which quantify the absolute amount of virus present in a sample in rapid time. This article will help you to learn more: What is the Ct value of SARS-CoV-2 (COVID-19) RT-PCR? 

Moreover, the qPCR is applicable in the Identification, characterization, genotyping and quantification of an infectious pathogen.  

Identification and quantification of circulating nucleic acid:

Cell-free fetal DNA and circulating micro RNA are two types of circulating nucleic acid present in the bloodstream. Both types of nucleic acids are present in very little quantity.

Even if it is very difficult to extract it, qPCR helps in the identification and quantification of circulating nucleic acid.

Circulating mRNA is the best marker for cancer diagnosis. Prenatal diagnosis of several inherited disorders is well characterized using real-time PCR.

Detection of pathogenic SNPs:

Melting curve analysis helps in the identification of many pathogenic SNPs, it is the most recommended method for characterization and identification of some of the diseases.

Apart from these applications, real-time PCR is also used in forensic studies, evolutionary studies, mutation creation, fossil studies and in other applied fields.

Read more:

• What is a multiplex PCR?
• What is a hot-start PCR?
• What is touchdown (TD)-PCR?

Conclusion:

Real-time PCR has been provided. the significant value during pandemics or epidemics for sensitive, real-time and rapid detection of pathogens to reduce the mortality and morbidity rate. Although, traditionally it has been used for tuberculosis diagnosis.

Read more: What is TB (Mycobacterium Tuberculosis) PCR?

Quantitative real-time PCR is an accurate, fast, sensitive, cheap and adequate method in genomic research. The diagnostic value of real-time PCR is more than any other PCR technique.

Sources:

Robert E (2010). RT-PCR: A Science and Art form. RNA methodologies 4th Edition, Academic Press, 385-448.