Real-time PCR, or qPCR, is a powerful tool for detecting and measuring DNA or RNA with unprecedented accuracy, which, unlike regular PCR, needs no gel electrophoresis for results visualization, and therefore enables quick turnaround, minimal contamination risk, and easier workflow.
What makes qPCR different is its ability to monitor the amplification process in real-time, displaying exponential curves on the computer. For RNA analysis, reverse transcription involves the transformation of RNA into DNA before being amplified. It is widely used in research and diagnostics, from studying plant viruses like TMV and Citrus Tristeza to detecting harmful bacteria.
qPCR can either be qualitative or quantitative, depending on the target pathogen or gene expression measurement scenario. Types of sequencing may range harmoniously depending on the genes studied, but for that matter, some relevant background knowledge is required in the case of the genes involved.
In research, medicine, agriculture, or forensics, it is a reliable and multipurpose tool that will propel advancements in the science of diagnostics.
Real-time PCR (qPCR) presents various advantages over standard PCR and other conventional nucleic acid detection methods, rendering it a more suitable platform for many laboratory applications.
One of its major strengths is the high quantitative range of nucleic acids that can be measured more than or equal to five log units. It can detect even less than five copies of the target molecule, or in some cases, down to a single-copy detection. This is especially helpful in working with limited samples, such as clinical biopsies or laser capture microdissection lysates. When paired with competent internal control and calculation procedures, qPCR assures highly reproducible results with a precision of as low as 1-2%, rendering it suitable for the analysis of minute changes in gene expression.
Unlike conventional PCR which derives quantification during the plateau phase of amplification, the practical big difference is that qPCR measures amplification with real-time detection as it happens during the exponential phase. The quantification approach derives from the continuous monitoring of the PCR process in real time minimizing problems with plateau effects and fluctuations in the assay.
One of the key advantages of qPCR technology could have been fast throughput; the whole process, from amplification to analysis of closed reaction, eliminates post-PCR manipulations such as gel electrophoresis. The result is substantially less work, less time, and less chance of the risk of requisite cross-contamination. Conventional PCR technique involves gel electrophoresis or probe hybridization to identify products of interest and is thus time-consuming, less automated, and less specific than qPCR.
Additionally, qPCR systems train faster and claim suitable conditions for high-throughput applications; therefore, they are appropriate for today's labs. qPCR allows for real-time visualization of the accumulation of specific products through every cycle, making the technique independent of external detection methods, reducing errors, and ideally increasing overall efficiency.
•Gene Expression Analysis: Real-time PCR is a cornerstone tool for gene expression studies across various research projects. Its ability to measure mRNA levels quantitatively enables researchers to study gene regulation under different conditions.
•Genotyping Models: Useful for genotyping knockout, knockin, and transgenic mouse models to understand genetic functions and phenotypes.
•Gene Knockdown and Delivery: Evaluate the efficiency of gene knockdown techniques and delivery methods in cell cultures or animal models.
•Single Nucleotide Polymorphism (SNP) Detection: Facilitates allelic discrimination to identify SNPs that may predispose individuals to diseases, aiding in epidemiological studies.
•Viral Load Monitoring: Tracks change in viral load, helping to measure disease progression and evaluate antiviral therapies.
•Bacterial Detection: Enables rapid and precise identification of bacteria such as Mycobacterium tuberculosis, Listeria monocytogenes, and Neisseria gonorrhoeae, leading to targeted antibiotic therapies and reduced use of broad-spectrum antibiotics.
•Antibiotic Resistance Monitoring:Mutation analysis through melting curves helps track antibiotic resistance in pathogens like Staphylococcus aureus and Helicobacter pylori.
•Oncology Applications:
a) Detects chromosomal translocations and fusion gene transcripts, aiding in the detection of minimal residual disease (MRD) and monitoring disease progression in leukemia.
b) Determines DNA copy numbers linked to malignancy and evaluates gene expression in solid tumors using small specimens (e.g., fine-needle aspirates).
•Time and Cost Efficiency:Provides faster diagnostic results (e.g., reducing pathogen analysis time from days to minutes) with high sensitivity compared to traditional methods.
•Detects genetically modified organisms, microbes, and parasites in food and agricultural products.
•Gene Expression Kinetics:Analyzes changes in gene expression in response to drug treatments.
•Drug Metabolism Studies:Evaluates the activity of transporters and enzymes involved in drug distribution and elimination.
•Real-time PCR's sensitivity and specificity make it ideal for analyzing limited or degraded samples in criminal investigations.
•Technological Advancements:Machines are becoming faster, cheaper, and more portable due to improvements in microfluidics, optics, and thermocycling. This will increase accessibility to real-time PCR globally.
•Global Access:Cost reductions and portability will enable applications in remote areas, supporting disease diagnosis and epidemiological studies.
•Education:Integrating real-time PCR into biology education can improve hands-on learning, encouraging students to pursue careers in science.
•Emerging Fields:Real-time PCR is expected to support expanding fields like proteomics, metabolomics, and transcriptomics, offering a more comprehensive understanding of biological systems.
By combining precision, speed, and versatility, real-time PCR has become an essential tool across multiple disciplines, with ongoing advancements promising even broader applications.
Understanding the Mechanism of Real-Time PCR
Real-time PCR (qPCR) analysis is often illustrated using an amplification plot, where the x-axis represents the number of PCR cycles, and the y-axis shows the fluorescence emitted from the amplification reaction, which correlates with the quantity of the amplified product.
The amplification plot can be divided into two main phases: the exponential phase and the plateau phase. In the exponential phase, the PCR product roughly doubles with each cycle. However, as the reaction progresses, the components of the reaction (such as primers, nucleotides, and polymerase) start to deplete. When one or more of these components becomes limited, the reaction slows down and enters the plateau phase, typically around cycles 28–40.
At the beginning of the reaction, fluorescence remains at background levels, and no detectable increase occurs despite the exponential accumulation of the PCR product (usually during cycles 1–18). Eventually, enough product accumulates for the fluorescence signal to become detectable. The point at which fluorescence surpasses the background is known as the quantification cycle (Cq). Since the Cq occurs during the exponential phase, when reagents are still abundant, it allows for an accurate and reliable estimation of the initial amount of template DNA or RNA in the reaction based on the known exponential kinetics.
The Cq value is primarily influenced by the initial template concentration. A high amount of template at the start of the reaction means fewer cycles are needed to generate a detectable fluorescence signal, resulting in a low or early Cq. Conversely, a low initial template concentration requires more cycles to reach detectable fluorescence, resulting in a high or late Cq. This relationship between template concentration and Cq is the basis for the quantitative aspect of real-time PCR.
Sample Collection
To accurately quantify gene expression, it is crucial to collect samples that are as homogeneous as possible. If the sample contains various cell types, identifying the expression patterns of the target gene could be challenging. For heterogeneous samples, methods such as tissue dissection, needle biopsies, or laser capture microdissection can be employed to isolate specific cell types. The collected cells can then be used for RNA extraction.
RNA Extraction
For real-time RT-qPCR, either total RNA or poly(A+) RNA is typically used. A major consideration when working with RNA is preventing the degradation of samples by RNases. RNase-free solutions are commercially available, or solutions can be treated with diethylpyrocarbonate (DEPC) and autoclaved to eliminate RNases. Labware can also be treated with DEPC or baked at 250°C for 3 hours to inactivate any residual RNases.
When preparing RNA samples, it may be necessary to perform DNase treatment to remove any genomic DNA contamination, as its presence can lead to an overestimation of the mRNA quantity. However, if the RNA sample is limited, DNase treatment should be approached with caution, as it may cause loss of the RNA. To avoid amplification of genomic DNA, it is essential to design primers that are specific to the transcript, such as primers that span across splice junctions.
Analyzing Nucleic Acid Quantity and Quality
Accurate quantification of nucleic acids is crucial for gene expression analysis, especially when RNA amounts are used to normalize target gene expression levels. RNA concentration and purity are typically assessed by measuring the ratio of UV absorbance at 260 nm and 280 nm. This ratio helps determine the quality and quantity of RNA samples before further analysis.
In summary,
Real-time PCR (qPCR) has revolutionized the field of molecular biology by offering a precise and efficient method for quantifying nucleic acids. Its ability to provide real-time monitoring of amplification allows for the accurate measurement of gene expression levels, enabling detailed insights into biological processes. The key to this technique lies in the relationship between the quantification cycle (Cq) and the initial template concentration, providing a reliable way to assess the abundance of target nucleic acids.
To ensure the success of real-time PCR experiments, proper RNA isolation, handling, and contamination prevention are crucial steps. With the correct sample collection, RNA extraction, and quality control, researchers can obtain highly reproducible and accurate results. Real-time PCR's sensitivity and versatility make it an indispensable tool in fields ranging from gene expression analysis to diagnostics, ultimately driving advancements in biomedical research, clinical diagnostics, and therapeutic development.