RT-qPCR, or Reverse Transcription quantitative Polymerase Chain Reaction, is a molecular biology method used to detect and measure specific RNA molecules within a sample. It serves as a tool for understanding gene activity and identifying the presence and quantity of RNA-based pathogens. RT-qPCR combines reverse transcription and quantitative PCR, converting unstable RNA into a more stable DNA form for amplification and detection.
The Need for Reverse Transcription
Standard Polymerase Chain Reaction (PCR) amplifies DNA sequences. However, many biological targets, such as messenger RNA (mRNA) or the genetic material of RNA viruses, exist as RNA. RNA molecules are less stable than DNA and cannot be directly amplified by PCR.
To overcome this, RNA targets must first be converted into a complementary DNA (cDNA) copy. This cDNA then serves as the template for the subsequent PCR amplification. This initial conversion, known as reverse transcription, enables the study of RNA-based genetic information using DNA-based PCR technology.
The Reverse Transcription Process
The reverse transcription process begins with an RNA template, such as mRNA, isolated from a biological sample. Reverse transcriptase, an enzyme, synthesizes a DNA strand from this RNA template.
The reaction mixture also includes primers and deoxynucleotide triphosphates (dNTPs). Primers are short DNA sequences that bind to the RNA template, providing a starting point for the enzyme. The dNTPs are the building blocks that the enzyme incorporates to synthesize the new complementary DNA strand. This creates a single-stranded cDNA molecule, which is then used for the quantitative PCR step.
The Quantitative PCR Process
Once cDNA is synthesized, it proceeds to the quantitative Polymerase Chain Reaction (qPCR) phase. qPCR measures DNA amplification in real time. Each PCR cycle consists of three main steps: denaturation, annealing, and extension.
Denaturation involves heating the mixture to 95°C, separating double-stranded cDNA into single strands. Annealing lowers the temperature, allowing primers to bind to specific regions on each single-stranded cDNA template. During extension, a DNA polymerase enzyme synthesizes new DNA strands by adding dNTPs, extending from the primers. This process doubles the amount of target DNA in each cycle.
qPCR is quantitative and real-time due to fluorescent chemistries that detect accumulating DNA products. Fluorescent dyes, such as SYBR Green, bind to double-stranded DNA, emitting a signal that increases with DNA production. Sequence-specific fluorescent probes, like TaqMan probes, bind to target DNA and release a signal upon degradation during amplification. The fluorescence signal’s intensity is directly proportional to the amplified DNA.
A qPCR instrument monitors this fluorescence. The Cycle threshold (Ct) value is the point where the fluorescence signal crosses a predetermined threshold, rising above background noise. This Ct value is inversely related to the initial amount of target DNA (and thus the original RNA). A lower Ct value indicates that the fluorescence signal was detected earlier in the reaction, meaning there was a higher initial quantity of the target molecule in the sample. Conversely, a higher Ct value suggests a smaller initial amount of target RNA was present, requiring more cycles to reach the detection threshold.
Understanding the Results
Interpreting RT-qPCR results revolves around the Cycle threshold (Ct) value. For example, a Ct value of 15-20 signals a high concentration, while values around 35-40 suggest a lower concentration or minimal presence. The relationship between the Ct value and the initial quantity of the target is linear on a logarithmic scale.
Researchers use standard curves for absolute quantification, comparing unknown samples to a reference curve to determine their exact copy number or concentration. Relative quantification methods, such as the comparative Ct method, determine fold change in gene expression between samples, often by normalizing to a stable internal control gene. These methods allow for comparisons of gene activity or pathogen levels across various experimental conditions or patient samples.
Key Applications of RT-qPCR
RT-qPCR is a technique used across various scientific fields due to its sensitivity and accuracy. A primary application is measuring gene expression levels. By quantifying messenger RNA (mRNA) transcripts, researchers determine how much a specific gene is “turned on” or “off” under different conditions, such as drug treatment or in different cell types. This provides insights into cellular processes and disease mechanisms.
The technique is also used for detecting and quantifying viral loads, making it a tool in diagnostics. For instance, RT-qPCR became a standard method for identifying and measuring SARS-CoV-2 virus in patient samples during the COVID-19 pandemic. Its ability to detect low copy numbers of RNA molecules makes it valuable for early detection of pathogens, including bacteria and fungi, in clinical, veterinary, and food safety settings. RT-qPCR is also applied in validating findings from other high-throughput methods like microarrays and RNA sequencing, and in studying RNA interference (RNAi).