Rapid qPCR Service, Digital PCR Service For Multiplex Gene Expression, Vector Copy Number, Residual DNA, Etc

PCR stands for Polymerase Chain Reaction. This technique was first developed in the 1980s, and since then, it has been used to generate a large amount of DNA from a small DNA volume.

PCR is a technique in itself. However, researchers often employ PCR in other processes, such as gel electrophoresis, DNA sequencing, and pathogen detection. PCR is fundamentally a diagnostic method used in different domains, including microbiology, virology, mycology, parasitology, and dentistry.

PCR amplifies a specific section of DNA. The process begins with a DNA template. An enzyme, polymerase, copy this piece of DNA. The PCR reaction has a heating and cooling cycle. Researchers employ this cycle to separate the DNA strand and make multiple copies of the same strand. The final assay reaction thus contains a large number of identical DNA copies that researchers can use for further analysis, such as Southern blotting and sequencing.

Quantitative PCR (qPCR) is similar to traditional PCR, but it can quantify the amount of DNA in the sample. qPCR analysis involves probes and fluorescent dyes that bind to the target DNA. These probes, when excited by a light source, emit different wavelength lights.

Researchers measure the wavelength of each emitted light to determine the amount of target DNA present in the sample. Besides, one may monitor DNA amplification in the actual moment by determining the increase in fluorescence in real time.

Reverse-transcriptase PCR (RT-PCR) is a PCR sub-type that generates cDNA from template mRNA molecules. RT-PCR can detect and quantify RNA viruses. The first step in RT-PCR analysis is reverse transcribing the mRNA template using reverse transcriptase enzyme. This cDNA is then amplified using PCR. RT-PCR is a sensitive and accurate technique to detect RNA viruses present at very low levels.

qPCR can measure gene expression on its own. In qPCR expression analysis, a section of DNA is read to synthesize the mRNA, which then produces the protein of interest. However, in RT-PCR, reverse transcriptase creates cDNA, which is then used in PCR to calculate the mRNA transcripts.

Droplet Digital PCR (ddPCR) is an advanced version of qPCR and qRT PCR analysis. ddPCR gene expression can identify and amplify specific gene sequences for subsequent quantification. While qPCR has been a gold standard for evaluating gDNA and cDNA levels, it has limitations when it comes to assessing small differences in gene expression. ddPCR gene expression reduces these limitations by employing a partition in the traditional qPCR analysis. This ddPCR method divides a typical qPCR run into thousands of droplets. Each droplet is in the nanoliter range. Hence, ddPCR gene expression determines the target molecules based on Poisson Distribution. Such accurate analysis has made ddPCR gene expression the primary technique in the absolute quantitation of copy number variation and rare mutations.

Digital PCR (dPCR) assay is a novel technique for measuring and identifying nucleic acids that can quantify them precisely and detect rare variants. It works by dividing and isolating a DNA or RNA sample into many separate, parallel PCR reactions; some of these reactions have the target molecule (positive) and others do not (negative). Here, a single DNA or RNA molecule can be amplified by a factor of a million or more. During amplification, TaqMan® Assays with dye-labeled probes bind to specific sequences to identify targets. When the target sequence is absent, no signal is produced. After PCR analysis, the proportion of negative reactions is used to calculate the exact number of target molecules in the sample, without requiring standards or internal controls.

Digital PCR analysis has many uses and is getting increasingly deployed to:

• Determine the genetic makeup of an organism
• Absolute quantification of DNA and RNA targets
• Perform copy number analysis (CNV)
• Detect rare mutations
• Identify pathogens, their strains, and their viral load
• Analyze proteins with assays that use proximity ligation

Some differentiating features of the QuantStudio™ Absolute Q™ Digital PCR dPCR System include:

• 90-minute turnaround time for quick answers
• ≥95% sample utilization, <5% dead volume to enable accurate quantitation
• Ease of use—complete one hands-on step with an entire plate in less than 5 minutes; no rolling or sticky seals
• Flexibility to run 4, 8, 12, or 16 samples per plate, saving consumables and lab resources
• Image subtraction allows debris and defects to be subtracted during analysis to improve calling accuracy for rare target

The primary difference between the two methods is that qPCR measures in real-time. This real-time feature means researchers can monitor target DNA amplification as it is happening.

Secondly, qPCR employs probes or fluorescent dyes. These probes quantify the total amount of target DNA present in the study sample.

Finally, the main function of PCR analysis is to amplify DNA and help in other downstream applications. On the other hand, qPCR is employed widely to detect and quantify RNA viruses.

In gene expression analysis, qPCR assays can determine the amount of mRNA present in the sample. A small mRNA region is amplified, while the qPCR tool measures the intensity of individual probes. As the number of cycles increases, the fluorescent signals increase with an increase in amplicons.

The Ct value represents the primary qPCR experience. The threshold in qPCR analysis is in the linear phase, and the curve crossing the threshold is the measured Ct value. The threshold value is unique for each qPCR assay.

The primary principle of qPCR analysis is that at each assay cycle, the PCR products double in number. Often, qPCR analysis gives a relative expression, which varies in the gene expression of the assay sample. Researchers may also determine the absolute quantification of a gene. However, absolute quantification requires a standard, which is generally the cloning of cDNA into a vector.

Real Time-PCR/qPCR quantification can be absolute or relative. Absolute quantitation determines unknown samples by interpolating their copy number relative to those obtained from a standard curve. Absolute quantitation may be used to study the correlation between viral or gene copy numbers and disease state. It is important to understand the exact copy number of the target RNA in a biological sample to be able to study the progress of the disease. Relative quantitation is used to analyze how the activity of genes in a sample is increased or decreased relative to a reference, control sample that has a normal or baseline gene activity. Relative quantitation is used for comparisons of healthy and diseased subjects, for determining genetic or phenotype related differences between samples., or to compare disease or disease treatment sample biomarkers to baseline levels. Both methods involve normalization of gene quantitation by simultaneous or parallel measurement of a housekeeping gene (for eg. beta actin or GAPDH) – the expression levels of which are relatively stable and are not altered by disease state or treatment. This ensures that differences in absolute or relative level of the gene changes observed are from disease state or treatment and not from differences in nucleic acid loading.

There are two approaches to qPCR analysis: dye-based and probe-based. Let us dive deep into each of these qPCR types.

Dye-based qPCR assays

This method measures the fluorescent signals, specifically the binding of the dye to the double-stranded DNA. Initially, the dye shows a weak signal. However, after binding to the double-stranded DNA, the signal increases dramatically. Hence, target sequence amplification increases the fluorescence, which directly relates to the amount of double-stranded DNA in the sample. This approach needs only two primers specific to the sequence. Hence, dye-based qPCR is a rapid way to assess a large number of study samples.

However, dye-based methods can detect all double-stranded DNA present in the sample. This non-specific detection can include primer dimers and non-specific products, leading to inaccurate results. Hence, reaction specificity is verified using the denaturing/melting curve after each qPCR run. Such an approach ensures that only the target DNA is amplified. Moreover, another limitation of this approach is that dye-based qPCR can only quantify one target sequence at a time.

Probe-based qPCR analysis

Probe-based qPCR measures the fluorescent signal of sequence-specific fluorophore-labeled probes. This method is more specific than a dye-based approach and hence is preferred in diagnostic applications.

Probe-based qPCR is a multiplexed method that can quantify multiple targets in a single sample reaction. This multiplexing approach employs a specific fluorophore for each target. Probe designs vary among experiments. However, hydrolysis probes are most commonly used in probe-based qPCR.

qPCR is a rapid and sensitive tool for the accurate determination of nucleic acids in diverse biological samples. Their applications include cancer phenotyping, gene expression analysis, and the detection of GMOs in food.

In research capacities, qPCR can provide quantitative analysis of gene copy numbers and the presence of mutant genes. Besides, qPCR can be combined with RT-PCR to quantitate alterations in gene expression. For example, researchers can monitor changes in mRNA levels to assess the effect of drugs and environmental conditions on gene expression levels.