Reverse Transcription Quantitative Polymerase Chain Reaction, or RT-qPCR, is a laboratory technique used to detect and measure the amount of a specific RNA molecule in a biological sample. This method combines two molecular biology techniques: reverse transcription and quantitative polymerase chain reaction. Its primary function is to provide a precise measurement of RNA levels, allowing scientists and medical professionals to understand genetic activity or identify the presence of RNA-based pathogens.
The Reverse Transcription Step
The initial phase of RT-qPCR addresses a limitation: the Polymerase Chain Reaction (PCR) component can only amplify DNA sequences. Many biological entities, such as certain viruses like coronaviruses and influenza, store their genetic information as RNA. Cellular activities also involve RNA molecules, which carry genetic instructions from DNA to guide protein production. To overcome this, the target RNA must first be converted into a DNA form.
This conversion is performed by an enzyme known as reverse transcriptase. Reverse transcriptase uses the RNA molecule as a template to synthesize a complementary DNA strand, often referred to as cDNA. This cDNA molecule then serves as the template for the subsequent amplification steps.
The Quantitative PCR Step
Following the creation of cDNA, the quantitative PCR step begins, which involves amplifying specific DNA sequences and measuring them in real-time. The newly synthesized cDNA is mixed with components, including DNA polymerase, specific primers, and fluorescent probes. This mixture then undergoes repeated cycles of heating and cooling in a specialized thermocycler instrument. Each cycle involves denaturing the DNA by heating to separate the strands, annealing primers to specific regions of the target sequence as the temperature cools, and extending new DNA strands using DNA polymerase.
Primers are short, synthetic DNA sequences designed to bind specifically to the beginning and end of the target cDNA sequence, defining the region to be copied. As new DNA strands are synthesized during each cycle, the amount of target DNA effectively doubles. Fluorescent probes are also present in the reaction mixture; these probes contain a fluorescent dye and a quencher molecule. When the probe is intact, the quencher suppresses the dye’s fluorescence, but as the DNA polymerase extends the new strand, it displaces and cleaves the probe. This separation of the dye from the quencher allows the dye to emit a fluorescent signal, which is detected by the RT-qPCR instrument.
The term “quantitative” in qPCR signifies that the machine measures the fluorescent signal during each amplification cycle. The intensity of this signal directly correlates with the amount of amplified DNA present in the reaction. This real-time measurement capability distinguishes qPCR from traditional PCR, which only provides an end-point detection. Continuous monitoring allows for precise quantification of the initial RNA amount.
Interpreting RT-qPCR Results
The data generated from an RT-qPCR experiment provides insights into the initial amount of RNA present in the sample. A key metric derived from this real-time measurement is the Cycle threshold, or Ct value. The Ct value represents the specific cycle number at which the fluorescent signal from the amplifying DNA crosses a predetermined threshold, indicating a detectable level of the target sequence. This threshold is set above the background noise but within the exponential phase of amplification.
A lower Ct value signifies that the fluorescent signal reached the threshold earlier in the amplification process, indicating a larger amount of the target RNA was present in the original sample. Conversely, a higher Ct value suggests more amplification cycles were needed to generate a detectable signal, implying the initial sample contained a smaller quantity of the target RNA. For diagnostic purposes, scientists establish a specific Ct cutoff value to determine whether a sample is considered “positive” or “negative” for a particular RNA molecule.
Common Applications of RT-qPCR
RT-qPCR is an important tool across various scientific and medical fields due to its sensitivity and specificity. One of its most prominent applications is in the diagnosis of infectious diseases. It is a primary method for detecting the presence of RNA viruses in patient samples. For example, RT-qPCR gained widespread recognition for its rapid and accurate detection of the SARS-CoV-2 virus, which causes COVID-19. It is also routinely used to identify other RNA-based pathogens, including influenza viruses and Human Immunodeficiency Virus (HIV).
Beyond diagnostics, RT-qPCR plays an important role in biological research, particularly in gene expression analysis. Researchers use this technique to quantify the activity levels of specific genes within cells or tissues. For instance, by comparing gene expression profiles in cancerous cells versus healthy cells, scientists can identify genes that are over- or under-expressed, providing insights into disease mechanisms. This information can guide the development of new therapeutic strategies and improve our understanding of complex biological processes.