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 dilution of known template concentrations can be used to establish a standard curve to determine the initial starting amount of the target template in experimental samples or to assess the efficiency of the reaction. For that concentration (y-axis) the log of each known concentration in the dilution series (x-axis) is plotted against the value of Ct. Details about the reaction efficiency and different reaction parameters (including slope, y-intercept, and correlation coefficient) can be obtained from this basic graph. The concentrations selected for the standard curve should encompass in the experimental samples the target’s expected concentration range.

Correlation coefficient (R2)

The correlation coefficient is a calculation of how well the standard curve matches the results. The R2-value represents the normal curve linearity. Ideally, R2 = 1, but the highest value usually is 0.999.

Absolute quantification

The sort of analysis that you select and the quantification relies on the initial goal. Total quantification lets you assess rates of gene expression in exact copy numbers.

Relative quantification

Relative quantification is very different, but still relies on a standard curve or curves being generated. Use the same samples, this form of quantification is used to evaluate fold differences in expression between two samples and is converted to a housekeeping gene.

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

There are three simple methodologies widely used to identify RNA or DNA targets by real-time PCR and all use fluorescent dyes. In each case, a small initial fluorescent signal is raised proportionally in parallel with the exponential rise in the product(s) of DNA produced during each successive PCR process.

  • The simplest assay method involves a free dye being incorporated into the freshly developed double-stranded DNA component. The most widely used dye in real-time PCR for this reason is SYBR ® Green I. The down side is that any double-stranded molecule formed in the reaction can generate a signal, such as priming dimers or incorrect PCR products.
  • There are several very common signaling schemes dependent on dye-primers required for real-time PCR. They vary from the very basic LUX (light upon extension, Invitrogen) primers to externally and internally quenched more complicated primers to the very complex framework of scorpion primers. Dye-primer-based assays enable multiplexing with SYBR ® Green I which is not feasible.
  • Promega’s Plexor device is a modern primer-based assay method that is now coming onto the market. The reason for this test is the usage of two cytodine and guanine, iso-C, and iso-G bases isomers. The iso-bases can only shape a base pair with the complementary iso-base and DNA polymerases can only add an iso-base while the complementary iso-base of the cognate is available in the current series. One primer is synthesized with an iso-C labelled fluorescently on the 5′ edge. The PCR master mix comprises free iso-dGTP combined with a dark quencher coloring (DABSYL).

Dye differentiation

Many PCR reactions in real time involve several dyes, like one or two reporter dyes, a quencher dye in certain situations, and, most frequently, a passive reference dye. Many dyes may be calculated separately in the same tube, either through tailored variations of excitation and emission filters, or through a method called multicomponenting.

Multicomponenting is a statistical procedure for the calculation of color strength for increasing reaction dye. Multicomponenting offers the advantages of easy dye designation error correction, refreshing optical performance to factory standard without hardware adjustment, and provides a source of troubleshooting data.

 Passive reference dyes

In real-time PCR, passive reference dyes are commonly used to normalize the fluorescent signal of reporter dyes and to fix fluorescence variations that are not PCR dependent. Standardization is required to fix differences from well to well induced by shifts in the concentration or volume of the reaction, and to fix variations in instrument scanning. Most real-time PCR instruments use ROX ™ dyes as the passive reference dye, because ROX ™ dye does not affect the real-time PCR reaction and has a fluorescent signal that can be distinguished from that of many reporter or quencher dyes used.

RT-PCR dyes (Fluorophore) emission and excitation

Real-time PCR assay types

Gene expression profiling is a popular application of real-time PCR, which measures the relative abundance of transcripts to establish variations of gene expression across samples. RNA accuracy, reverse transcription performance, real-time PCR output, quantification strategy and selection of a standardizer gene play especially important roles in experiments on gene expression.

Assays for calculating the viral titer may be difficult to build. Scientists also seek to measure the amount of viral copies in the samples. This is often done by comparing a standard curve produced using known genome equivalents or nucleic acid extracted from control of a titered virus. Quality relies on the accuracy of the material used to construct the regular curve. Reverse transcription and real-time PCR performance also play major roles, depending on the type of the aim — an RNA or DNA virus —. Whether the assay s for counting functional viral particles or the total number of particles will also be influenced by assay design.

In copy number variation analysis, the genome is analyzed for duplications or deletions. The design of assays, and more importantly the standard generation of curves, would be determined by whether relative or absolute quantification is necessary. Assay architecture focuses on PCR performance in real-time and the precision required to distinguish differences in a single sample.

Finally, assays of allelic discrimination will identify differences down to the stage of single nucleotides. Unlike the approaches mentioned above, fluorescence of endpoints is calculated to establish the genotypes of the SNP. The architecture of the primers and probes plays especially important roles to ensure a low occurrence of cross-reactivity common to allles.

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

There always rises a confusion when using short acronym to refer PCR. To alleviate this, it’s called Quantitative Real Time PCR (qPCR) in Real Time. However the method of synthesizing cDNA from RNA is Reverse Transcriptase. It is named Reverse Transcriptase quantitative polymerase chain reaction (RT-qPCR) when conducting qPCR from the RNA specimen.

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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