Real-Time PCR (qPCR) – Definition, Principle, Protocol, Application, Advantages

What is Real-Time PCR?

• Real-Time PCR (also called quantitative PCR or qPCR) is a variation of the Polymerase Chain Reaction which allows the progress of DNA amplification to be monitored during each cycle, rather than only at end point.
• The fluorescence signal is used as reporter, which increases proportionally with the amount of PCR product (amplicon) present in reaction, and is measured in real time.
• The method employing fluorescent dyes (for example SYBR Green) or sequence-specific probes (for example TaqMan) is used, depending on requirement of specificity and sensitivity.
• The starting quantity of nucleic acid (DNA or RNA after reverse transcription) is inferred by cycle number at which fluorescence crosses a defined threshold (Ct or Cq), fewer cycles indicating greater initial template.
• Advantages are provided by Real-Time PCR in terms of sensitivity, speed, dynamic range, and reduced risk of contamination since no post-PCR manipulation (eg gel electrophoresis) is needed.
• When RNA is target, reverse transcription is performed first to convert RNA into complementary DNA (cDNA) then amplification / detection by qPCR is done.
• The instrument used must have capability of fluorescence detection and thermal cycling, so that fluorescence data can be collected after each cycle.
• Applications are many (diagnostics, gene expression quantification, detection of pathogens etc), because Real-Time PCR allows both qualitative (presence/absence) and quantitative (copy number, relative expression) analysis.

Definition of Real-Time PCR

Real-time PCR, also known as quantitative PCR (qPCR), is a molecular biology technique that allows for the amplification, detection, and quantification of DNA or RNA in real-time during the amplification process. It uses fluorescent dyes or probes to measure the increase in fluorescence intensity, providing quantitative data on the amount of target nucleic acid present in the sample.

Principle of Real-Time PCR – How Does Real-Time PCR Work?

The Principle of Real-Time PCR is based on amplification of target DNA (or cDNA) by Thermal Cycling, while fluorescence is monitored continuously, so that product accumulation is detected during each cycle.

With inclusion of Reporter / fluorescent dye (non-specific dyes like SYBR Green, or sequence-specific probes such as TaqMan), the fluorescence signal increases in proportion to the amount of PCR product produced, and this increase is measured cycle by cycle.

The conversion of RNA into complementary DNA (cDNA) is performed first, when RNA is to be analysed, because DNA polymerase cannot amplify RNA directly; then the PCR amplification proceeds using cDNA as template.

During each cycle, three main temperature-dependent steps are performed: denaturation (strand separation), annealing (primer binding), extension (new strand synthesis by DNA polymerase), as in conventional PCR; but with the added detection stage via fluorescence after or during the extension or probe cleavage step.

A threshold fluorescence level is defined, above background noise, and the cycle number at which fluorescence crosses this threshold (Cq or Ct value) is used to infer the initial quantity of target nucleic acid, because earlier crossing indicates larger starting template.

Amplification efficiency is assumed (or must be validated) to be approximately constant during exponential phase, so that doubling (or near doubling) of product per cycle occurs until reaction components become limiting, then plateau is reached.

The instrument (thermal cycler with optical-detection module) is equipped to both change temperature rapidly for each PCR step, and detect fluorescence (via detector / camera etc) in a closed-tube format, so that signal is collected without opening reaction vessels (which reduces contamination).

Melting curve / dissociation curve analysis can be applied after amplification when non-specific dyes are used, to verify specificity of the product, by observing at which temperature double-stranded DNA melts (dissociates) which reflects product sequence purity.¹²³

Steps of Real-Time PCR (Protocol)

1. At first, RNA is extracted from cells / tissues, which must be purified and free of contaminants such as proteins, genomic DNA, RNases.
2. After extraction, RNA integrity is checked, often by spectrophotometry (A260/A280) or agarose gel / Bioanalyzer, so that only good quality RNA is used.
3. Genomic DNA is removed, by DNase treatment or other methods, so that amplification of contaminating DNA is avoided in downstream steps.
4. Reverse Transcription (RT) is performed, in which RNA is converted into complementary DNA (cDNA), using Reverse Transcriptase enzyme, suitable primers (gene-specific / random / oligo-dT), dNTPs, buffer, and co-factors.
5. The RT reaction is incubated at appropriate temperature (often ~42-50°C) for required time (for example 30-60 min), so that reverse transcription proceeds; then RT is inactivated by heat if needed.
6. Real-Time PCR mix is prepared, by combining cDNA template, forward and reverse primers, DNA polymerase (thermostable), fluorescent reporter (dye or probe), buffer, Mg2+ etc. Master mix may be prepared to reduce pipetting variation.
7. Thermal cycling is performed: denaturation, annealing, extension steps repeated for many cycles (often ~35-45), with fluorescence measurement at each cycle.
8. Fluorescence detection is done in each cycle (either during extension or after), via a dye (eg SYBR Green) or probe (eg TaqMan), so that accumulation of PCR product is monitored in real time.
9. Threshold is set above background fluorescence, and cycle number at which fluorescence crosses threshold (Ct or Cq) is determined, which is used for quantification of initial template amount.
10. Controls are included: no-template control (NTC) to check for contamination, no-RT control to check for genomic DNA contamination, positive control / housekeeping gene to check assay performance.
11. After amplification, when using non-specific dyes, melting curve (dissociation curve) analysis is performed, so that specificity of product is verified (to detect primer dimers or non-specific amplification)
12. Data are analysed, Ct / Cq values are compared across samples, efficiencies may be checked, normalization done (with internal control genes), relative or absolute quantification is performed.

The working procedure of Real-Time PCR

The working procedure of Real-Time PCR is generally divided into two broad steps, and these steps are amplification and detection.

A. Amplification

• Denaturation – At very high temperature (usually around 95 °C), the double-stranded DNA is separated into single strands, and secondary structures are loosened. The maximum temperature which the DNA polymerase can withstand is usually chosen, although when GC content is high, the denaturation time is sometimes increased for better efficiency.
• Annealing – In this step, the primers hybridize with complementary sequences. An appropriate temperature is selected, usually about 5 °C lower than the melting temperature (Tm) of the primers, so that specific binding occurs.
• Extension – At temperature near 70–72 °C, DNA polymerase shows its optimal activity, and primer extension is carried out, often at a rate of almost 100 bases per second. In cases where the amplicon is very small, the extension step is sometimes combined with annealing step at around 60 °C, which reduces cycle time.

B. Detection

• Fluorescence principle – The detection part is carried out with fluorescence technology, which is added in the reaction in the form of dyes or probes.
• Thermal cycling – The specimen is placed into the proper wells and subjected to thermal cycles, like in conventional PCR, but here, the detection is simultaneous.
• Light source – A tungsten or halogen source is applied by the Real-Time PCR machine, which excites the marker dye, and fluorescence is produced in direct proportion with copy number increase.
• Signal emission – The emitted fluorescence is captured by the detector, which then converts the optical signal into digital form. After that, data is sent into the computer system.
• Threshold crossing – The signal is observed on a screen, and when fluorescence crosses a defined threshold level (minimum detection level of the detector), the sample is considered positive, and the cycle number of that crossing is recorded (Ct value).⁸

Real-time PCR assay types

Gene expression profiling assays – In such assays, the relative abundance of transcripts is assessed, and expression patterns between different samples are determined. RNA quality, reverse transcription efficiency, and real-time PCR efficiency are considered crucial. The strategy for quantification is selected, and a normalizer gene is chosen, because normalization ensures reliability of comparison. Gene expression assays are widely used but they require careful optimization, since minor variations in cDNA synthesis may produce large changes in calculated expression.

Viral titer determination assays – These assays are designed to quantify viral copy number within samples. Quantification is usually performed by comparison with a standard curve, which is generated using known genome equivalents or nucleic acid extracted from a titered virus control. The reliability of such assays is dependent on the accuracy of the material used in curve generation. When the target is an RNA virus, reverse transcription must be included before PCR, and the efficiency of both steps affects results. The Assay design is also influenced by whether the aim is to quantify functional infectious virus, or total viral particles present.

Genomic profiling assays – In these assays, genomic DNA is examined for duplications or deletions. The design depends strongly on whether relative or absolute quantification is intended, because this determines how the standard curve is constructed. A major focus is placed on the efficiency of real-time PCR, and the precision required to discriminate even single-copy differences in the genome. For many experimental systems, this requirement for accuracy makes optimization challenging and sometimes repetitive.

Allelic discrimination assays – These are employed to detect genetic variation, even at the level of a single nucleotide polymorphism (SNP). Unlike the previously described assays, the endpoint fluorescence is measured instead of the continuous monitoring across cycles. The information obtained is used to determine the zygosity status of a genome. Primer and probe design have critical importance, because allele-specific cross-reactivity must be minimized to avoid misleading signals. In such assays, sensitivity and specificity both are highly dependent upon probe chemistry.

What is one-step RT-PCR?

• The One-step RT-PCR method is a protocol in which reverse transcription (RT) and polymerase chain reaction (PCR) are combined in a single reaction tube, in a common buffer, with both reverse transcriptase and DNA polymerase present.
• The RNA is converted into complementary DNA (cDNA) by the reverse transcriptase, and amplification of that cDNA is effected by the DNA polymerase, all without transferring material between tubes.
• The One-step format is chosen when speed, simplicity, or high-throughput are required, because fewer pipetting steps are involved, workflow is simplified, and risk of contamination is reduced.
• Gene-specific primers are required in One-step RT-PCR, since RT and PCR are in same reaction, and non-specific priming (eg oligo(dT) or random hexamers) may not be compatible with PCR conditions that follow.
• It is less flexible than Two-step RT-PCR in terms of target number per sample, because only those targets for which primers are included can be amplified in same tube.
• The sensitivity or efficiency may be somewhat lower in some scenarios, because reaction conditions must compromise between optimal RT and optimal PCR phases.
• The method is advantageous when sample RNA is limited or when fewer genes are to be measured, as material loss is minimized, and turnaround time is shortened.
• In One-step RT-qPCR (quantitative version), the above process is coupled with a detection system (fluorescent probes or dyes) so that the amplified product is measured in “real time” as amplification proceeds.¹⁴

What is Two-step qRT-PCR?

• Two-step qRT-PCR is a technique in which the reverse transcription (RT) and the quantitative PCR (qPCR) are performed in separate reactions.
• The RNA first is transcribed into complementary DNA (cDNA) by reverse transcriptase, using primers such as oligo(dT), random hexamers, or gene-specific primers.
• Then, an aliquot of that cDNA is used as template in a separate real-time PCR reaction to amplify specific target sequences and quantitate them.
• The flexibility in primer choice for the RT step is provided, which allows for broad transcript coverage (when random/oligo(dT) primers are used), or specific targeting (when gene-specific primers are employed).
• Greater sensitivity is often achieved, especially for low abundant transcripts, because each reaction (RT and qPCR) can be optimized independently.
• A stable cDNA pool is generated, which can be stored for later use, or used in multiple qPCR reactions for different targets from the same original RNA sample.
• Disadvantages include increased handling steps, which may raise risk of contamination, more time being required, and more complexity in workflow.
• Use-cases are provided mostly where multiple genes are to be quantified from one RNA sample, or when assay sensitivity / flexibility is needed (eg, challenging sequences or unknown targets).¹³

Differences Between one-step vs two-step RT-PCR

Fluorescence Markers used in Real Time PCR

• The Types of fluorescence markers are broadly divided into DNA-binding dyes (non-specific) and sequence-specific probes, which differ in specificity, cost, design complexity.
• The DNA-binding dye (such as SYBR Green I) is used, which binds to any double-stranded DNA (dsDNA) in the reaction; when bound fluorescence increases strongly, and signal reflects total dsDNA present (target + non-specific products).
• The TaqMan probe chemistry is used, which employs an oligonucleotide probe labelled with a fluorescent reporter at 5’ end and quencher at 3’ end; probe hybridizes to target, and during extension the probe is cleaved by the polymerase’s 5′→3′ nuclease activity, releasing reporter from quencher, signal being produced only if correct target is present.
• Molecular Beacons are used, which are hairpin-shaped probes, with a fluorophore and quencher at ends; fluorescence is quenched when probe is in stem-loop form but restored when probe hybridizes to target sequence, because stem loop opens.
• The Scorpion probes / LUX / Eclipse / hybridization (LightCycler-type) probes are also used, which employ different designs of probe / primer combinations to get specificity, sometimes using fluorescence resonance energy transfer (FRET) or quencher/reporter separation upon binding or extension.
• The Reporter dyes commonly used include FAM, VIC, TET, etc, which differ in excitation/emission spectra, and are used in dual-labelled probes like TaqMan probes.
• The Quencher moieties are included in probe-based systems, which suppress reporter fluorescence until the probe is cleaved or binds target. Examples include TAMRA, DABCYL, or dark quenchers like Black Hole Quencher (BHQ) that do not emit light, minimising background.
• Passive reference dyes are used in many master mixes, which help normalize well-to-well / cycle-to-cycle variability of fluorescence that is not due to amplification (optical differences, pipetting etc).
• dsDNA-binding dyes are cost-effective / simpler design, because only primers are needed, but specificity is lower, and melting-curve analysis is often required to verify correct amplicon.
• With probe-based markers, high specificity is achieved, because only target-specific probe binding or cleavage yields fluorescent signal, reducing false positives coming from primer-dimers or non-target amplifications.⁵⁴⁶⁷

Applications of Real-Time PCR

• The Gene expression (mRNA) analysis is performed, because changes in transcript levels under different conditions (drug treatment, stress, developmental stages etc) are quantitated.
• Genetic variation analysis / mutation detection is done, which allows identification of single nucleotide polymorphisms (SNPs), insertions / deletions, or point mutations in genes.
• Pathogen detection and quantification is enabled, because Real-Time PCR can detect (and measure) viral, bacterial, fungal nucleic acid in samples with high sensitivity.
• Diagnostic testing is facilitated, as screening / monitoring of infectious diseases, and genetic disease markers can be done using real-time PCR, often for early detection / disease progression / therapeutic monitoring.
• GMO (Genetically Modified Organism) detection is carried out, because specific transgene or modification sequences are identifiable and measurable in crop / food / environmental samples
• Allelic discrimination and genotyping is applied, so that different alleles in a population or sample are distinguished, which is important in studies of variation or pharmacogenomics.
• MicroRNA and noncoding RNA analysis is supported, because even small non-coding RNA transcripts can be reverse transcribed and quantified, providing insight into regulatory RNAs.
• Copy number variation (gene dosage) is measured, which is beneficial in research where gene amplification / deletion is significant (eg oncology) since gene copy changes impact phenotype.
• Environmental / ecological studies are aided, since microbial load or species abundance can be quantified in environment or food / soil / water samples.
• Monitoring of treatment or therapeutic efficacy is conducted, because real-time PCR allows viral load / pathogen load to be tracked over time, for assessing response to therapy.
• Mutation / variant tracking is executed, especially in epidemiology, so that emerging variants in pathogens (virus variants etc) or mutational shifts are identified.
• Quality control in biomanufacturing or diagnostics is insured, because real-time PCR is used to check purity, contamination, or presence of unwanted nucleic acids.⁹¹⁰¹¹

Advantages of Real-Time PCR

• The Dynamic range of detection is broad, which enables quantification across many orders of magnitude (eg 6-8 logs), so both low and high abundance targets are measured precisely.
• High sensitivity is provided, because small amounts of template (DNA or RNA) are detectable, and low copy number targets can be found.
• Quantification is done during exponential phase, which yields more accurate / reliable data than endpoint (plateau) detection, because exponential amplification is less influenced by reagent depletion and non-linear effects.
• The initial copy number of target is allowed to be determined with accuracy, because Ct / Cq values are captured early in reaction.
• Reduced post-PCR handling is achieved, which lowers risk of contamination, because amplification and detection are performed in same closed tube system.
• Rapid turnaround of results is enabled, since gel electrophoresis or other end-point analyses are not required, which saves bench time / hands-on time.
• Multiplexing capability is supported, because multiple fluorescent dyes / probes can be used in same reaction, which allows simultaneous detection of several targets.
• Improved reproducibility is implied, because threshold cycles (Ct/Cq) and standard curves or internal controls are used, which help normalize variation across different runs / samples.
• Throughput is increased, as many samples / wells are processed automatically by real-time PCR machines, and data analysis is often software-assisted.
• Flexibility in qualitative / quantitative outcomes is allowed, because real-time PCR can be used to detect presence/absence of sequences, or to measure how many copies are present.
• Specificity is improved when sequence-specific probes are used, because non-specific signals (primer-dimers, off-target amplifications) are reduced, which improves signal clarity.¹²

Real-Time vs. Digital PCR vs. Traditional PCR

FAQ