Quantitative Reverse Transcription Polymerase Chain Reaction, or QRT-PCR, is a laboratory technique that measures the precise quantity of specific RNA molecules in a sample. This method quantifies gene expression levels or the presence of RNA viruses. Its “quantitative” nature provides numerical data on RNA abundance, unlike methods that only confirm presence.
The Reverse Transcription Step
RNA molecules cannot be directly amplified by standard Polymerase Chain Reaction (PCR) because PCR enzymes require a DNA template. To overcome this, QRT-PCR begins with a reverse transcription step. An enzyme called reverse transcriptase converts the target RNA into a complementary DNA (cDNA) strand. This cDNA then serves as the stable template for the subsequent PCR reaction.
Primers, short DNA sequences, guide the reverse transcriptase to synthesize the cDNA. The amount of cDNA produced directly correlates with the initial RNA quantity in the sample. The efficiency of this conversion impacts the accuracy of the final quantification.
Real-Time Amplification and Detection
Following reverse transcription, the newly synthesized cDNA undergoes amplification in the “PCR” part of QRT-PCR. This involves repeated cycles of DNA synthesis, duplicating the cDNA template exponentially. Each cycle consists of denaturation, annealing, and extension, rapidly increasing target DNA copies. The “real-time” aspect allows continuous monitoring of DNA amplification as it occurs, differentiating it from traditional PCR.
During amplification, fluorescent molecules are incorporated into the reaction. These molecules, such as DNA-binding dyes or specific probes, emit light when they bind to the newly synthesized DNA. As amplified DNA increases with each cycle, the fluorescent signal intensity also increases. A specialized instrument detects this accumulating fluorescence, providing a real-time readout.
The point where the fluorescent signal crosses a predetermined threshold is the threshold cycle (Ct value). This value is inversely proportional to the initial amount of target RNA. A lower Ct value indicates a higher initial RNA quantity, as fewer amplification cycles were needed to reach the detection threshold. This real-time measurement allows for precise quantification, unlike traditional PCR’s end-point analysis.
Where QRT-PCR is Used
QRT-PCR is widely used across scientific and medical fields due to its precision and sensitivity in RNA quantification.
One application is in detecting and quantifying viral loads, such as monitoring HIV or SARS-CoV-2 infections. Measuring viral RNA in a patient’s sample helps clinicians assess disease progression and treatment effectiveness.
The technique is also used in gene expression studies to measure specific gene activity within cells or tissues. Researchers quantify messenger RNA (mRNA) levels to understand how genes respond to different conditions, like development, drugs, or diseases such as cancer. This aids in identifying biomarkers and understanding molecular mechanisms.
In diagnostics, QRT-PCR assists in early and accurate infectious disease diagnosis. Its ability to detect minute amounts of pathogen RNA makes it a tool for identifying infections before symptoms appear. This sensitivity supports public health surveillance and disease outbreak control. The method also supports new therapy development by precisely measuring their impact on gene expression or pathogen levels.