What is real-time PCR?

Posted by Jack on December 8, 2022

Using real-time PCR (RT-PCR) to analyze gene expression is a powerful tool. However, there are limitations to its use.

Quantification strategies in real-time PCR

Choosing the most appropriate quantification strategy is the first step to ensuring accurate interpretation of qPCR data. The most common PCR quantification strategy, relative quantification, provides information about changes in gene expression in relation to another reference gene. However, this method can be inaccurate and result in incorrect interpretations. A more precise and faster quantification method, absolute quantification, can be used to detect mRNA expression levels.

Absolute quantification of mRNA levels can be achieved by using a calibration curve. This is a linear relationship between the amount of input DNA and the amplicon size. Ideally, a calibration curve should contain an interval between the GOIs, or targets, that is measured in the quantification assay. It should be able to identify false positive up- or downregulation that are not caused by real amplification. In addition, it allows researchers to quantify multiple genes simultaneously.

Traditional methods for PCR quantification relied on non-specific intercalating dyes. Although these probes can be used in many situations, they are not always suited for use in qPCR assays. They are also subject to variations of technical origin, which can lead to results that are unreliable. The introduction of AccuCal addresses some of these problems, and it can now be integrated into qPCR experiments.

In this study, the PCR assay was evaluated for detection of CNS viruses. The accuracy of absolute quantification was assessed in eight independent research groups. The range was from 92 in to 501 bp, covering the range of amplicon sizes typically used in qPCR. The R2 value for the linear calibration curve was 0.9987. The slopes for the two amplification products (AccuCal-D and AccuCal-P) were 0.9653 and 0.9601, respectively. The values for the dilution series were indistinguishable. This suggests that AccuCal-D was an effective way to perform absolute quantification of RT-qPCR in dye-based master mixes.

Several reference genes can be used in qPCR experiments. These genes are usually included in gene expression studies, but they can vary with the tissue, experimentation conditions, and RNA concentration. If a single reference gene is used, the uncertainty interval can be larger. Nevertheless, several reference genes can be used, and the relative quantification model can be easily adjusted to accommodate the variability of these reference genes.

Alternatively, researchers can choose to perform absolute quantification by constructing a standard curve. This approach provides linearity of the quantification range, and it is based on the same principles as relative quantification. The amplicon size and the primers are critical in determining the specificity of a PCR assay. The quantity of amplicons that are amplified increases with each cycle of amplification. In addition, the amplification process includes efficiency corrections. This allows researchers to obtain the efficiency of amplification for each sample within an assay.

The AccuCal method is easier to apply than the conventional relative quantification methods. It is also cheaper. This method can be used to measure the same amplicon size in both qPCR and probe-based assays. The method also provides robust absolute quantification. This is important because steady-state mRNA levels may not be directly related to protein abundance.

Using real-time PCR to analyze gene expression

Using real-time PCR to analyze gene expression is an efficient and economical method for identifying gene activity. The process requires a combination of target-specific primers and probes that are designed to amplify target genes. The fluorescence emitted during amplification is related to the amount of the target present in the sample. These techniques are widely used to study the activity of genes and are considered the gold standard for gene expression quantification. It is a versatile and reproducible technique that is easily obtainable and is also low in cost.

The accuracy of qRT-PCR is affected by a number of factors. It depends on the amplification efficiency, cDNA quality, and the RNA integrity. Various reference genes are used to determine the expression stability of a gene. For example, BestKeeper is a software tool that uses pairwise correlations to determine the most stable housekeeping genes. It can identify the most suitable internal genes for qRT-PCR normalization.

In this study, we used several reference genes to validate the expression stability of the E and N gene of the SARS-CoV-2 virus. Our analysis included a comparison of their relative expression levels in the host cells of SARS-CoV-2 infected patients with healthy controls. The reference genes include ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, eukaryotic translation initiation factor 3E, actin, and top. The efficiency of the PCR assays was also calculated. The results showed that the amplification efficiency was over 110% for the top and actin genes.

A comprehensive ranking based on four algorithms was used to select the most suitable internal genes. The resulting list includes UBC2 and CYP. Varshney RK also validated the housekeeping genes.

A comparison of the transcriptional response of the three target genes of SARS-CoV-2 was performed. The transcriptional profile reveals the transcription factors that regulate the target genes and helps scientists compare their transcriptional responses. The transcription factor regulating a particular gene is called the elongation factor and can help scientists understand how that gene is regulated. A knockout of this factor can be used to characterize new transcription factors. This technique is useful for scientists looking to discover a specific transcription factor or compare the activity of several transcription factors.

We used a combination of real-time RT-PCR and quantitative display PCR to detect and quantify cytokines, RDRP, N, and E of the SARS-CoV-2 virus. The RT-PCR assays were carried out in a 96-well microplate with an optical film. The results were analyzed using GraphPad Prism 8 software. The amplification curve of the housekeeping gene was also determined. We then compared the transcriptional profiles to the corresponding amplification curves for each of the target genes to determine which genes had been differentially expressed in the SARS-CoV-2 infected cells. The amplification curves of the housekeeping gene were examined for statistical significance. The results indicated that the amplification curves of the housekeeping genes were relatively smooth.

Limitations of real-time PCR

Despite the advantages of real-time PCR, there are a few limitations that can lead to inaccurate results. Several factors must be considered when designing a PCR assay. These include primer specificity, primer stability, and amplification efficiency.

Using the correct primers for a target gene is very important. Incorrect primers can result in false positive results and poor results overall. Choose the best primers for your PCR by choosing conserved regions, examining exon location, and selecting primers that are not susceptible to amplification interference.

Amplification efficiency is also a critical factor in amplification and the overall results. It is necessary to have a high amplification efficiency to ensure accurate data. However, small variations in thermal cycling conditions or mispriming events can result in large changes in amplified product. Fortunately, this variation can be offset with an endogenous control. An endogenous control is a nucleic acid that is present in the individual sample. An ideal control gene is stable and expressed in a constant manner regardless of the experimental conditions.

When designing a PCR assay, it is crucial to identify the phenotypic and biochemical characteristics of bacterial strains. This is important to determine whether a PCR assay is applicable to your specimen. If not, you can opt for alternative methods such as classic PCR. Traditional PCR techniques are sensitive and reproducible, but they may lack the specificity to distinguish between different RNA sequences.

Having the right tools to perform real-time PCR is essential. Several in-silico tools are available to aid in the analytical design of PCR experiments. These tools include Q-Gene, an automated software application that allows you to perform various statistical tests to calculate expression values. This software is especially helpful when you are dealing with multiplexed targets. It can significantly speed up the data processing of your PCR experiment.

Real-time PCR assays are designed to detect gene expression differences between samples as low as 23%. This is a significant improvement over conventional quantitation techniques. In addition, these assays are capable of increasing sample throughput and have a lower coefficient of variation. Moreover, they are easier to use and require less reagents. The use of reagents can lead to contamination, but this is usually less common in real-time PCR.

There are two main steps to a PCR assay: isolation of the viral genome and amplification of the resulting product. When using a standardized method, you can obtain accurate results with 100% amplification efficiency. Depending on the threshold height, you can also obtain false-positive results.

In clinical laboratories, real-time PCR is used to detect a variety of viruses. This method is especially useful for monitoring treatment outcomes. Using a real-time PCR assay to analyze food allergens is another example of its effectiveness. The use of probe-based real-time PCR assays has greatly improved the diagnosis of viral infections.

Other applications of PCR are to differentiate between different pathogens and mutations in pathogen genes. These are used in research and development to identify new and emerging diseases. Although they are more expensive than traditional methods, PCR assays can provide rapid, high-throughput, and accurate results.

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