Gene expression, the process by which information from a gene is used in the synthesis of a functional gene product like a protein or RNA, is a foundational biological mechanism. Understanding the levels at which specific genes are expressed provides deep insights into cellular function, disease states, and responses to various stimuli. Quantitative Polymerase Chain Reaction, or qPCR, offers a precise and widely adopted method for accurately measuring these gene expression levels. This technique allows researchers to quantify nucleic acids with high sensitivity across a broad range of applications.
Understanding qPCR Fundamentals
qPCR, also known as real-time PCR, is a laboratory technique that amplifies and simultaneously detects DNA. The core principle involves monitoring the accumulation of DNA during the reaction in real-time, rather than just at the end. This real-time detection is achieved through the use of fluorescent molecules, such as DNA-binding dyes or specialized probes, which emit light as more DNA copies are produced. As the DNA amplifies exponentially, the fluorescent signal increases proportionally, allowing for quantification of the initial amount of target DNA.
The Concept of Relative Expression
Relative expression involves comparing the amount of a specific target gene in one sample against its level in another, such as in a treated cell sample versus an untreated control, or a diseased tissue versus a healthy one. To ensure accurate comparisons, a process called normalization is applied. This accounts for variations between samples, such as differences in the initial amount of starting material or the efficiency of the experimental procedures. A “reference gene,” often referred to as a housekeeping gene, is used for this normalization because its expression levels are expected to remain relatively constant across different experimental conditions and samples. Common examples of such genes include GAPDH, ACTB, or 18S ribosomal RNA.
How Relative Expression qPCR is Performed
The process begins with collecting biological samples, such as cells or tissues, ensuring uniformity. RNA is isolated from these samples, with care taken to prevent degradation by RNases. High-quality RNA is converted into complementary DNA (cDNA) through reverse transcription, using reverse transcriptase. This cDNA serves as the template for the qPCR reaction, which is set up by combining it with specific primers for the gene of interest and the chosen reference gene, along with DNA polymerase and fluorescent detection reagents. These reaction mixtures are then placed into a real-time PCR instrument, which controls the temperature cycles necessary for DNA amplification and measures the increasing fluorescence.
Interpreting Relative Expression Results
The raw data from a qPCR experiment are expressed as Ct (Cycle threshold) values, representing the PCR cycle number at which the fluorescent signal crosses a predetermined threshold. A lower Ct value indicates a higher initial amount of the target gene’s RNA, meaning the signal was detected earlier in amplification. To interpret relative gene expression, these Ct values are used to calculate “fold changes” between samples. This involves the comparative Ct method (ΔΔCt method), which determines the difference in Ct values between the target and reference genes, comparing this difference across experimental and control samples. A fold change value of 1 indicates no change in gene expression between the tested and control conditions. A fold change greater than 1 signifies an increase in gene expression, while a value less than 1 suggests a decrease; for example, a fold change of 2 means the gene’s expression has doubled, whereas a fold change of 0.5 means its expression has been halved.
Applications of Relative Expression qPCR
Relative expression qPCR is a versatile tool with broad applications across various scientific fields. In disease research, it is frequently used to study changes in gene expression linked to disease progression, identify potential biomarkers, and determine how specific genes are regulated in cancerous cells compared to healthy ones. The technique is also widely employed in drug development to evaluate the efficacy of new treatments by observing how they alter gene expression in cells or tissues. Beyond medicine, relative expression qPCR contributes to fundamental studies of gene regulation, allowing scientists to understand how genes are turned on or off in response to different biological cues. It is also valuable in validating gene knockdown or knockout experiments, confirming that a target gene’s expression has been successfully reduced or eliminated.