by Yasemin Cole
Technique Name: qRT-PCR (quantitative real-time polymerase chain reaction)
Fun rating: 3/5
Difficulty rating: 2/5
Purpose: In molecular and genetic biology, we oftentimes create experiments to uncover the inner workings of the molecular world. Unbeknownst to us, the millions of cells in our body perform complex tasks in a matter of milliseconds. One such process is the production of RNA, a temporary genetic code storage unit, which is translated into protein (following the central dogma of biology). Quantitative real-time polymerase chain reaction (qRT-PCR) is a technique that quantifies the level of RNA, a readout for gene expression, in a sensitive and precise manner.
How it works:
- To perform qRT-PCR, one must first collect the cells of interest and extract the RNA after the cells are lysed. Scientists typically use cell culture, or cells from a petri dish, as a model to study RNA expression.
- Using an enzyme called reverse transcriptase, the RNA from cells will be converted into complementary DNA (cDNA, the reverse step of the central dogma), a highly stable set of molecules. As you may recall, the nucleotide bases of RNA and DNA are similar with one exception (uracil bases are utilized in RNA while thymine bases are used in DNA). This process converts all of the RNA into cDNA. However, you may only be interested in specific proteins or genes.
- To quantify the levels of specific genes, fluorescent primers are used to amplify regions of interest, similar to a typical PCR reaction. As a refresher to PCR please read this blog post. When the fluorescent probes are incorporated into the growing strand, the machine detects the fluorescence and it is interpreted as the number of PCR products. As the machine cycles through each temperature, a new reading is taken and plotted onto a curve. As seen in the figure below, the fluorescent reporter is normally “quenched” such that the fluorescence is not detected by the machine. However, when it is cut by polymerase (which normally adds nucleotides to the growing strand of DNA), the fluorescent probe lights up and the fluorescence is quantified by the detector.
Application:
A researcher may be studying a gene and is curious whether a mutation leads to low levels of RNA expression. For example, mutations that occur at the beginning of the gene can cause small fragments of RNA to be produced and subsequently degraded. Thus, the researcher may use qRT-PCR to quantify the level of RNA expression. One of the gold standard tests for COVID-19 infection utilizes this technology. As you may know, COVID-19 is a coronavirus that utilizes RNA in its genetic material. To quantify the amount of RNA in an individual’s body (such as from a nasal swab), the RNA is converted into cDNA, amplified, and quantified through qRT-PCR.
