Reverse Transcription quantitative Polymerase Chain Reaction, or RT-qPCR, is a laboratory technique used to analyze genetic material. It precisely detects and measures specific genetic information within a sample, combining two molecular biology tools for accurate insights into biological processes.
Measuring Gene Activity
RT-qPCR primarily measures the quantity of specific RNA molecules within a sample. RNA, or ribonucleic acid, serves as an intermediary molecule that carries genetic instructions from DNA to guide protein production. The amount of a particular RNA molecule in a cell or tissue reflects the activity level, or “expression,” of the corresponding gene. Higher RNA quantity indicates a more active gene, while lower quantities suggest reduced activity.
This technique detects and quantifies RNA from various sources, including infectious agents like viruses. For example, RT-qPCR played a role in detecting the genetic material of the virus causing COVID-19, as well as other pathogens such as HIV or influenza. RT-qPCR provides a quantitative understanding of gene activity or pathogen load.
The Reverse Transcription Step
The “RT” in RT-qPCR stands for Reverse Transcription, the initial step of the process. RNA molecules cannot be directly amplified in the subsequent Polymerase Chain Reaction (PCR) stage because PCR works with DNA templates. Therefore, RNA must first be converted into a DNA form.
This conversion is carried out by an enzyme called reverse transcriptase. This enzyme uses the RNA molecule as a template to synthesize a complementary DNA strand, known as complementary DNA or cDNA. The resulting cDNA is a stable copy of the original RNA sequence, making it suitable for amplification and quantification.
The Quantification Step
Following reverse transcription, the newly synthesized cDNA is amplified using the “qPCR” part of the technique, quantitative Polymerase Chain Reaction. This process increases the number of specific cDNA copies. qPCR measures this amplification in real-time.
To achieve real-time measurement, fluorescent dyes or probes are incorporated into the reaction mixture. As more DNA copies are generated during each cycle, these fluorescent molecules emit light. A specialized machine continuously monitors this increasing light intensity. The point where the fluorescent signal crosses a set detection threshold is called the “cycle threshold” or Ct value. A lower Ct value indicates the target RNA was initially present in a higher amount, as fewer amplification cycles were needed to reach the threshold.
Real-World Uses
The measurement capabilities of RT-qPCR make it useful across many scientific and medical fields. In disease diagnosis, it is routinely used to detect and quantify viral or bacterial genetic material in patient samples, aiding in identifying infections and monitoring disease progression.
RT-qPCR also plays a role in cancer research by measuring gene activity levels in tumor cells. This helps scientists understand how cancer develops, progresses, and responds to treatment. In drug development, the technique assesses how new medications affect gene expression, providing insights into a drug’s mechanism and efficacy. In basic biological research, RT-qPCR is a tool for studying how genes are turned on and off under different conditions.