Understanding Real-Time Quantitative Reverse Transcription PCR (qRT-PCR)

Real-time Quantitative Reverse Transcription PCR (qRT-PCR) is a crucial evolution of PCR technology that enables the reliable detection and measurement of products generated during each cycle of the PCR process. This technique has revolutionized the field of molecular biology by providing a direct, quantitative, and time-efficient method to analyze RNA levels.

What is Real-Time Quantitative Reverse Transcription PCR?

Real-Time Quantitative Reverse Transcription PCR, or qRT-PCR, is a technique that combines reverse transcription (RT) of RNA into DNA and the amplification of specific DNA targets. It is predominantly used to measure the amount of a specific RNA by monitoring the amplification reaction using fluorescence—a technique collectively referred to as real-time PCR or quantitative PCR (qPCR).

From RT-PCR to qRT-PCR: A Brief Overview

The term "real-time" in RT-PCR can be misleading, as it often leads to the abbreviation RT-PCR, which can cause confusion with another commonly used technique, reverse transcription PCR (RT-PCR). True RT-PCR refers to the process of first transcribing RNA into complementary DNA (cDNA) through reverse transcription and then amplifying the cDNA using PCR.

Applications and Advantages of RT-PCR and qRT-PCR

RT-PCR has become widely used for several reasons:

it does not require post-PCR processing,

it can measure a wide range of RNA abundance,

it provides both qualitative and quantitative insights.

Due to its simplicity, specificity, and sensitivity, RT-PCR is utilized in a multitude of applications, from simple experiments such as quantifying yeast cells in wine to more complex uses like detecting infectious agents such as the avian flu virus.

The Significance of RT-PCR Kits

The quality and purity of RT-PCR kits are paramount for achieving successful experimental results. Leading Biology offers RT enzymes engineered to efficiently create cDNA from various challenging RNA samples, ensuring high-quality and thermostable first-strand synthesis results.

Standard PCR vs. RT-PCR and qRT-PCR

PCR (Polymerase Chain Reaction) is a widely used molecular biology technique to amplify and detect DNA or RNA sequences. The standard PCR process consists of three main steps: denaturation, annealing, and extension, which are repeated over several cycles.

RT-PCR allows the use of RNA as the starting material by employing reverse transcriptase to synthesize cDNA from RNA. This cDNA can then be used as a template for either standard PCR or quantitative PCR (qPCR) reactions.

Quantitative PCR (qPCR): A Step Forward

Quantitative PCR (qPCR), a subcategory of RT-PCR, enables the precise measurement of nucleic acid concentration in real time. Unlike standard PCR, which only enables the detection of the presence or absence of a target sequence, qPCR allows for the quantification of the starting quantity of the target DNA.

Fluorescent-based Detection in qPCR

In qPCR, fluorescent dyes such as SYBR green bind to DNA. This binding allows for the measurement of fluorescence at each cycle of the qPCR, which increases proportionally with the amount of replicated DNA. Data is typically collected during the exponential phase of the reaction, providing quantitative information on the starting quantity of the target DNA. The fluorescence intensity is plotted against the cycle number to generate an amplification plot.

Amplification Plot and Parameters

The amplification plot shows two distinct phases: an exponential phase followed by a non-exponential plateau phase. During the exponential phase, the amount of DNA doubles with each cycle. As the reaction progresses, reagents are consumed, and ultimately, one or more components become limiting, leading to a plateau phase. The cycle at which this limiting occurs is defined as the threshold cycle (Ct), which is determined primarily by the initial amount of the target DNA. A large starting amount results in a lower Ct number, while a smaller starting amount results in a higher Ct number.

Applications of qRT-PCR

qRT-PCR is primarily used in various fields including gene expression analysis, allelic discrimination, and genetically modified organism detection. Its ability to provide both qualitative and quantitative information makes it increasingly popular for a wide range of research and diagnostic applications.

Conclusion

Real-time quantitative reverse transcription PCR (qRT-PCR) has significantly advanced our ability to analyze RNA levels, providing a powerful tool for various research and diagnostic applications.