Real-Time PCR

Basics of PCR

During a number of amplification cycles, real-time PCR collects fluorescent signals from reactions in the polymerase chain. Quantitative real-time PCR involves converting the fluorescent signals from reactions into a numerical value for each sample.

PCR is normally based on a quantitative relationship between the quantities of target sequence present at the start and the quantity of PCR product amplified at any given cycle. A correlation of this characteristic follows an exponential trend, resulting in a doubling of the products created at each cycle. Because the reaction takes place in a closed environment, such an exponential step is absolutely confined to a certain number of PCR cycles. This situation depletes reactant concentrations, enzyme activity, and other factors while increasing products over time.

Thus, the PCR is specified with four reaction stages known as:

  • Baseline: A very short stage in which the amplification is still undetectable.
  • Exponential: The reaction kinetics contribute to a desirable doubling of amplicons.
  • Linear: Characterized by slowdown amplification trend and no longer doubles the products at each cycle.
  • Plateau: In fact, the reaction is terminated, no more amplicon accumulation is achieved even if the number of cycles is increased and PCR products may start to degrade, which is very unusable.

In the exponential process the fluorescence signal is proportional to the DNA concentration, real-time identification will take place.

Steps in Real-time PCR

Every cycle in a real-time PCR reaction consists of three major steps. In total, 40 loops of reactions take place.

  1. Denaturation: High temperature incubation is used to free the “fusion” of double-stranded DNA into single-stranded DNA. The highest temperature resistant to DNA polymerase (usually 95 °C) is oftenly used. If the GC content is high, the denaturation time can be extended.
  2. Annealing: As the complementary sequences are likely to be hybridized during annealing, an appropriate temperature based on the observed melting temperature (Tm) of the primers (usually 5 °C below the Tm of the primer) can be utilized.
  3. Extension: At 70-72 °C, DNA polymerase activity is at its optimum, and primary extension occurs at rates of up to 100 bases per second. If the amplicon in real-time PCR is small, this step is usually combined with the annealing stage at 60 °C.

Overview of real-time PCR components

DNA polymerase

Since PCR effectiveness is usually linked to thermostable DNA polymerase, enzyme selection is critical. One of the key factors that influences PCR specificity is the fact that Taq DNA polymerase has non-specific annealing of primer activity at low temperatures. As a result, the primer annealing to DNA produces a non-specific product. A “hot-start” enzyme can significantly reduce the issue of non-specific products caused by poor annealing. DNA polymerase is a thermally sensitive enzyme; it does not become active during the early reaction setup and DNA denaturation.

Polymerases used in PCR

Reverse transcriptase

The Reverse Transcriptase (RT) is just as important as the polymerase DNA in qRT-PCR results. It is essential to select an RT that not only produces high levels of cDNA but also performs well at high temperatures. High temperature efficiency is also highly important in RNA denaturation with secondary structures. In a single stage of qRT-PCR, the RT that conserves its activity at higher temperatures enables you to use a target-specific primer with high melting temperatures (Tm).

dNTPs

Choosing the dNTPs and thermo-stable DNA polymerase from the same manufacturer is a smart strategy. It is common for experiments utilizing reagents from different vendors to experience a significant decrease in threshold cycle (Ct).

Magnesium concentration

In real-time PCR, a final concentration of 3 mM of magnesium chloride or magnesium sulfate is often used. The concentration is sufficient for most applications. However, the optimal magnesium concentration ranges between 3 and 6 mM.

Template

In each real-time PCR experiment, 10–1,000 copies of the template nucleic acid are used. This is about equivalent to 100 pg to 1 μg of genomic DNA or 1 pg to 100 ng of total cDNA generated via RNA. Excess templates can potentially introduce larger levels of contaminants, reducing PCR efficiency significantly. Depending on how specific the PCR primers are for cDNA rather than genomic DNA, treating RNA templates may be necessary to limit the possibility of contamination with genomic DNA. To address the issue, ‘DNase I’ is an option.

Pure, intact RNA is required for high-quality, full-length cDNA synthesis and may be required for effective mRNA quantification. Any RNase exposure must be prevented, and aseptic conditions should be ensured.

PCR primer design

Effective primer design is a crucial process in real-time PCR. In general, primers should be 18–24 nucleotides in length. This delivers actual annealing temperatures. The primers should be designed in accordance with standard PCR principles. They will be target sequence specific and safe from secondary structure. Primers also need to prevent extended homopolymer sequences or repeating motifs from erroneously annealing. Primer pairs should have suitable melting temperatures (± 1°C) and contain about 50% GC content.

Experimental technique

To obtain precise reproducibility results (usually, triplicates), design a master mix that contain all of the reaction components except the sample. The use of a master mix reduces the number of pipetting steps, reducing the possibility of cross-well contamination and other pipetting errors.

Internal controls and reference genes

For normalizing qPCR readings, reference genes or housekeeping genes are being used. Many studies have shown that reference gene identification is necessary to assure consistent and efficient assays. Housekeeping genes are often part of a biochemical system that is essential to an organism’s survival and whose activity is constitutive, or not changed significantly in response to various environmental stimuli that regulate gene expression of interest.

Examples

AssaysReference Genes
Viral Reverse Transcription-PCRBacteriophage MS2, RNAse P
Most organisms ranging from Human to microbeGlyceraldehyde-3-phosphate dehydrogenase
Most organisms ranging from Human to microbe18 S rRNA

Real-time PCR analysis

Baseline

The real-time PCR reaction baseline is the signal intensity during the first PCR cycles, typically 3 to 15 cycles, when fluorescent signal fluctuations are low. The reaction history, or “noise,” may be compared to the low-level baseline signal. For each output in real-time PCR, the baseline is determined subjectively by user analysis or by the automated amplification plot’s analysis. The baseline must be properly specified in order to compute the threshold cycle (Ct) appropriately. The baseline evaluation will determine enough cycles to eliminate the data captured in the initial stages of amplification, but it will not discover the interval where the amplification signal begins to rise above normal.

Threshold

The real-time PCR reaction threshold is the proportion of signal that reflects a quantitatively major shift over the measured baseline signal. The amplification signal is differentiated from the baseline signal in this process. Normally, the PCR instrument algorithm automatically sets the threshold at 10 times the standard deviation of the baseline fluorescent value in real time. However, the threshold position can be set at any point throughout the PCR’s exponential phase.

Ct (threshold cycle)

The threshold cycle (Ct) is the number of times the reaction fluorescence signal crosses the threshold. The Ct value is utilized to calculate the starting quantity of DNA copies since it is inversely proportional to the speciemn quantity at the starting point. For example, a specimen with double the starting material does achieve a Ct one cycle earlier than a specimen with half the amount of its starting material. This also signifies that the PCR operates at 100% output in all reactions.

The PCR amplification is also template-oriented. The consumption of numerous templates produces a massive number of amplicons. As a consequence, the more the initial material, the earlier the multiplication and the lower the Ct value measured. Ct values are ~3.3 cycles apart in a 10-fold difference in template quantity.

Standard curve

A series of dilutions of known template concentrations can be used to build a standard curve to calculate the starting quantity of the target template in experimental samples or to analyze the efficiency of the reaction. The log of every known concentration in the dilution series (x-axis) is plotted against Ct for that concentration (y-axis). Such simple graphs may be used to get information about the reaction efficiency and other reaction characteristics (such as the slope, y-intercept, and correlation coefficient). The concentrations used in standard curve should also include the target’s expected concentration in the test sample.

Correlation coefficient (R2)

The correlation coefficient is a measure of how closely the standard curve fits the results. The R2-value denotes the linearity of the normal curve. R2 should ideally be 1, however the highest value is generally 0.999.

Absolute quantification

The analysis and quantification are reliant on the initial quantity of the test sample. Absolute quantification aids in determining the quantity of gene expression in exact copy numbers.

Relative quantification

Relative quantification is quite different, but somehow it relies on  standard curves. This type of quantification compares the gene expression of an unknown sample to that of other reference samples.

Typical qPCR plot (FAM Reporter). Red line indicates threshold. The initial amplification curve has the Ct value of 21 and the last amplification curve has ct value of 40.5

Real-time PCR fluorescence detection

Based on dye chemistry, there are two basic categories for identifying nucleic acid targets by real-time PCR. One method makes use of dyes that interact with double-stranded DNA. The other approach employs fluorescent probes that are specific to a sequence. The fluorescent signal increases in proportion to the exponential growth of the DNA products produced after each PCR amplification cycle.

DNA binding dyes

The fundament of the non-specific, real-time PCR detection method is DNA binding dyes. These catogery dyes function by interacting with the DNA strand’s minor groove. These dyes emit strongly when intercalating with the double stranded and subjected to light of a particular wavelength that corresponds to the dye but exhibit negligible fluorescence when free in solution.

Furthermore, it is very obvious that these minor groove binding dyes might make it difficult to interpret the results of PCR assays because they also interact with primer dimers and other non-specific amplifications. Primer-dimers frequently arise in situations when there are few or no specific templates.

Fluorescent based qPCR

Fluorescent probes

The specificity of PCR is enhanced by amplicon detection using aprobes. The simultaneous multiplexing of several targets is made possible by the employment of a fluorescent dye-infused probe that efficiently absorbs, emits, and releases energy at various wavelengths.

The signal to noise ratio refers to the relationship between a fluorescent dye signaling positive hybridization and a signal from aberrant fluorescence. It is preferable to have a high signal-to-noise ratio (i.e. more signal, less noise) In general, oligoprobe chemistries that use a non-fluorescent quencher and need the least interaction to produce a signal are most preferred.

Fluorescent probe based qPCR

Dye differentiation

The signal-to-noise ratio refers to the relationship between a fluorescent dye signaling positive hybridization and a signal from unwanted fluorescence. It is preferable to have a high signal-to-noise ratio (i.e. more signal, less noise). In general, oligoprobe chemistries that use a non-fluorescent quencher and need the least interaction to produce a signal are most appropriate for this.

Multicomponenting is a statistical method for determining color intensity as the concentration of reaction dye increases. Multicomponenting provides advantages including rapid and easy dye signal collection error correction, signal detection update without hardware adjustment, and providing a source of troubleshooting data.

RT-PCR dyes (Fluorophore) emission and excitation
RT-PCR dyes (Fluorophore) emission and excitation

Passive reference dyes

Passive reference dyes are often used in real-time PCR to normalize the fluorescent output of reporter dyes and to correct fluorescence fluctuations that are not PCR dependent. Standardization is essential to correct variances between wells caused by changes in the concentration or volume of the reaction, as well as to correct variations in instrument readings. Most real-time PCR equipment utilize ROX dyes as the passive reference dye because they do not interfere with the real-time PCR reaction and have a fluorescence signal distinct from that of many reporter or quencher dyes.

Real-time PCR assay types

Gene expression profiling is a common real-time PCR technique that evaluates the relative abundance of transcripts to identify differences in gene expression across samples. In gene expression assays, RNA accuracy, reverse transcription performance, real-time PCR output, quantification approach, and selection of a standardizer gene all play critical roles.

It may be challenging to develop assays for estimating viral titers. Scientists also want to know how many viral copies are in the samples. This is frequently accomplished by comparing a standard curve generated using known genome equivalents or nucleic acid isolated from a titered virus’s control. The accuracy of the materials used to create the standard curve determines reliability. Depending on whether the target is an RNA or DNA virus, reverse transcription and real-time PCR efficiency play important roles. Test design will also influence whether the assay counts functional viral particles or the overall number of particles.

The genome is examined for duplications and deletions in copy number variation analysis. The design of assays, and more critically, the generation of standard curves, would be decided by whether relative or absolute quantification is required. The assay architecture focuses on PCR performance in real-time and the accuracy necessary to detect variations in a single sample.

Allelic discrimination techniques may detect variations as small as single nucleotides. In contrast to the previous techniques, the fluorescence endpoint is estimated to determine the genotypes of the SNP. The architecture of the primers and probes is especially important in ensuring that allele-specific cross-reactivity is prevented.

Real time PCR (RT-PCR) and Reverse Transcriptase Quantitative PCR (RT-PCR)

When utilizing a short abbreviation to refer to PCR, there is always some ambiguity. To address this, Quantitative Real Time PCR (qPCR) in Real Time is used. However, Reverse Transcriptase is used to create cDNA from RNA. When qPCR is performed on an RNA specimen, it is referred to as Reverse Transcriptase quantitative polymerase chain reaction (RT-qPCR).

Troubleshooting qPCR

References:

  1. Understanding PCR, A Practical Bench-Top Guide. Academic Press
  2. RT-PCR Protocols 2nd Edition. Humana Press
  3. Real-Time PCR handbook by Applied Biosystems. Life Technologies
  4. Real-Time PCR by Taylor and Francis Group
  5. Quantitative Real-Time PCR. Methods and Protocols. Humana Press
  6. PCR. Methods and Protocols. Humana Press
  7. Molecular diagnostic pcr handbook. Springer

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