The Northern Blot is a foundational laboratory technique used in molecular biology to investigate gene expression by specifically detecting and quantifying RNA molecules within a sample. This method allows scientists to determine the rate at which a particular gene is transcribed into messenger RNA (mRNA). The procedure is a modification of the earlier Southern Blot, which analyzes DNA, and was named in 1977 by James Alwine, David Kemp, and George Stark. It remains a powerful tool for analyzing transcript levels and providing insights into the molecular state of a cell or tissue.
RNA Preparation and Separation
The Northern Blot protocol begins with the extraction of total RNA from a homogenized tissue or cell sample. Maintaining RNA integrity is paramount because ubiquitous ribonucleases (RNases) actively degrade RNA, necessitating the use of strong denaturing agents like guanidinium isothiocyanate during homogenization to inactivate these enzymes. Once the total RNA is isolated, it is separated through a process called gel electrophoresis.
RNA samples are loaded into an agarose gel, which acts as a sieve, allowing an electrical current to pull the negatively charged nucleic acid fragments through the matrix. Smaller RNA fragments migrate faster and further down the gel than larger fragments, resulting in a separation based on size. To ensure that the RNA molecules are separated by size alone and not by their complex three-dimensional structures, the electrophoresis is performed under denaturing conditions, typically by including formaldehyde in the agarose gel. Formaldehyde disrupts the hydrogen bonds that could cause RNA to fold into secondary structures, guaranteeing a linear separation.
Following electrophoresis, the quality of the separation can be visually assessed by staining the gel with a fluorescent dye and observing the distinct bands corresponding to the two main ribosomal RNA subunits (28S and 18S) under UV light. This visual check confirms that the RNA has not been degraded and is separated correctly before proceeding. The separated RNA fragments are still trapped within the fragile agarose matrix, which is unsuitable for the subsequent detection steps, necessitating their transfer to a more robust support material.
Transfer to Membrane
The next procedural phase involves transferring the size-separated RNA from the gel onto a sturdy, solid support membrane. This step is necessary because the large nucleic acid probes used for detection cannot effectively penetrate the agarose gel matrix. The membrane material is typically positively charged nylon or nitrocellulose, which provides a stable platform for the RNA to be immobilized.
The transfer, or blotting, is commonly achieved using capillary action, where a buffer solution is drawn up through the gel, carrying the RNA molecules with it onto the membrane placed directly above. An alternative, faster method is electroblotting, which uses an electric current to actively drive the RNA from the gel onto the membrane. In both cases, the spatial arrangement of the RNA fragments on the membrane precisely mirrors the separation pattern achieved in the gel.
After the transfer is complete, the RNA must be permanently attached, or “fixed,” to the membrane so it is not washed away during the subsequent hybridization steps. For nylon membranes, fixation is often achieved by exposing the membrane to short-wave ultraviolet (UV) radiation, which creates covalent cross-links between the RNA and the membrane material. Alternatively, protocols may use a baking step in a high-temperature oven to immobilize the RNA. The fixed membrane is then ready for the highly specific detection phase.
Detection Through Hybridization
Nucleic acid hybridization is the mechanism for identifying the target RNA sequence on the membrane. This process requires a detection tool known as a probe, which is a short, single-stranded piece of DNA or RNA that has a sequence exactly complementary to the target transcript. To make the probe traceable, it is chemically labeled with a reporter molecule, such as a radioisotope or a fluorescent tag.
The membrane is incubated with the labeled probe in a specialized hybridization buffer under precise temperature and salt concentration conditions. These conditions are finely tuned to allow the probe to anneal only to its perfect match—the target RNA sequence—while preventing non-specific binding to unrelated RNA or the membrane itself.
Following the hybridization period, the membrane is washed stringently to remove any unbound or loosely bound probe, ensuring that only the probe specifically hybridized to the target RNA remains. Visualization depends on the type of label used on the probe. Historically, probes were labeled with the radioisotope phosphorus-32, and the resulting signal was captured on X-ray film via autoradiography, producing a dark band where the target RNA is located.
Modern techniques often employ non-radioactive labels, such as biotin or digoxigenin, which are then detected using an enzyme linked to a chemical substrate that produces light, known as chemiluminescence. The intensity and position of the resulting bands, whether on film or captured by a digital imager, reveal the amount of the specific target RNA and its precise size.
Scientific Uses of the Northern Blot
The primary use of the Northern Blot technique is to measure and compare the relative abundance of a specific messenger RNA (mRNA) transcript across various biological samples. By quantifying the signal intensity of the detected band, researchers can determine if a particular gene is being expressed more or less actively in one condition compared to another. This application is valuable for studying how gene expression changes during different stages of development, in response to environmental stimuli, or between healthy and diseased tissues, such as cancer.
Beyond simple quantification, the technique’s ability to separate RNA molecules by size provides a unique advantage for molecular characterization. The precise location of the band on the blot indicates the length of the target transcript, allowing researchers to detect subtle differences in gene products. This size resolution can reveal the presence of alternative splice variants, where a single gene produces multiple different mRNA transcripts of varying lengths, or identify prematurely terminated transcripts.