Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) is a molecular biology technique that precisely measures genetic material. It is an important tool in various scientific fields, providing insights into biological processes and disease states. Its ability to quantify specific molecules makes it valuable in both research and clinical settings.
What qRT-PCR Quantifies
qRT-PCR quantifies specific RNA molecules in a sample, indicating how much of a particular gene is active. Measuring messenger RNA (mRNA) quantity directly shows cellular activity and gene expression levels. The technique also quantifies viral RNA, crucial for diagnosing infections and monitoring disease progression, as seen with viruses like HIV or SARS-CoV-2. Beyond mRNA and viral RNA, qRT-PCR can quantify microRNAs (miRNAs), small RNA molecules that regulate gene expression.
How qRT-PCR Works
The qRT-PCR process begins by converting RNA into complementary DNA (cDNA). An enzyme called reverse transcriptase facilitates this transformation, as the PCR step works with DNA. This initial step, known as reverse transcription, is the “RT” part of qRT-PCR.
Once cDNA is synthesized, it serves as a template for exponential amplification using the PCR process. During each PCR cycle, the amount of DNA doubles, rapidly increasing the target sequence. This amplification ensures that even small amounts of initial RNA can be detected and quantified.
The quantitative (“q”) aspect of qRT-PCR involves real-time detection of DNA amplification. Fluorescent dyes or probes emit a signal as more DNA is produced. A specialized instrument monitors this fluorescence cycle by cycle, allowing precise measurement of the initial RNA quantity based on when fluorescence crosses a set threshold.
Key Applications
qRT-PCR is widely used to diagnose and monitor various diseases. It detects and quantifies pathogens like viruses and bacteria, helping clinicians assess infection severity and track treatment effectiveness, including monitoring viral load changes.
In cancer research, qRT-PCR studies changes in gene expression within cancer cells. It identifies molecular markers linked to tumor development and progression, offering insights into disease mechanisms. The technique also supports the search for new biomarkers for early detection or prognosis.
The method is also used in drug discovery and development. Researchers use qRT-PCR to evaluate how new drug candidates affect gene expression in cellular or animal models. This helps understand a drug’s mechanism of action and its potential therapeutic effects.
Beyond these specific areas, qRT-PCR is a foundational tool in basic biological research, exploring gene function, cellular processes, and how cells respond to environmental changes.
Distinguishing qRT-PCR from Related Techniques
Many molecular biology techniques use the term “PCR.” Polymerase Chain Reaction (PCR) amplifies specific DNA segments, producing millions of copies from a small starting amount. This technique is primarily used for qualitative detection, indicating the presence or absence of a DNA sequence.
Reverse Transcription PCR (RT-PCR) converts RNA into cDNA before PCR amplification. While RT-PCR detects RNA molecules, it is qualitative or semi-quantitative, showing RNA presence but not exact quantity. Amplified products are often visualized at the end of the reaction, such as on a gel.
Quantitative PCR (qPCR), also known as Real-Time PCR, quantifies DNA directly during amplification using fluorescence. It measures the amount of DNA as it accumulates in real-time, providing accurate quantification of the initial DNA template.
Finally, qRT-PCR combines Reverse Transcription and quantitative Real-Time PCR. This allows precise measurement of RNA levels by first converting RNA to cDNA and then quantifying that cDNA in real-time.