Northern blot is a laboratory method that separates RNA molecules by size through gel electrophoresis, transfers them onto a membrane, and uses labelled probes to identify specific transcripts.
Explanation
The northern blot technique was developed in the late 1970s as an analogue to the Southern blot used for DNA. It is designed to detect specific RNA species within a complex mixture and to estimate their abundance and size. In a typical northern blot, total or poly(A)+ RNA is extracted from cells or tissues and denatured to prevent secondary structure. The RNA samples are then resolved by electrophoresis on an agarose gel under conditions that separate molecules based on length. Following electrophoresis, the separated RNAs are transferred to a nylon or nitrocellulose membrane through capillary or vacuum blotting. Once immobilized, the membrane is hybridized with a labelled DNA or RNA probe that is complementary to the target sequence. After washing away unbound probe, the pattern of hybridization is visualized by autoradiography or chemiluminescence. Northern blots reveal the size of the transcript and allow comparison of expression levels between samples. While newer techniques such as quantitative reverse transcription PCR and RNA sequencing provide higher sensitivity and throughput, northern blotting remains valuable for verifying transcript size, detecting splice variants, and assessing RNA integrity.
Applications and examples
Researchers have used northern blotting to map gene expression during development, such as measuring beta globin mRNA levels in fetal versus adult tissues. It is also employed to confirm alternative splicing events; for example, different isoforms of the dystrophin transcript can be resolved by their distinct sizes. Plant scientists have applied the technique to monitor expression of stress responsive genes under drought or cold conditions. In clinical laboratories, northern blots have been used to detect viral RNA in patient samples, although such applications have largely been replaced by PCR based diagnostics. Despite its decline in routine use, the method remains a reliable way to determine transcript length and validate high throughput data.
Northern blotting continues to provide a straightforward means of characterizing RNA species and remains a useful complement to more advanced sequencing methods.
Related Terms: Southern Blot, Western Blot, Quantitative RT PCR, RNA Hybridization, RNA Sequencing