Introduction
The first blotting technique developed by Dr. Edwin Southern is called the Southern blot. This technique was subsequently modified so as to detect other target molecules. These techniques being variations of Southern blot were named northern blot, western blot, and eastern blot. Northern blot is used to detect specific RNA sequences in a sample. Therefore it is also called the RNA blot.
Pimp your brain: Northern Blot
Principle
In northern blot, the sample RNA is separated on the basis of size with the help of gel electrophoresis. The separated RNA fragments are transferred to a support membrane and then treated with a labeled DNA probe. If the sample contains the complementary RNA sequence, the probe will bind to the membrane and it can be visualized using various methods.
Procedure
The key steps in the northern blot technique are as follows:
- The RNA sample is first separated by size using agarose gel electrophoresis. RNA molecules form streaks rather than bands on the gel as there are several small fragments of RNA.
- The RNA molecules are then transferred to a special blotting paper usually made of nitrocellulose. Membranes made up of nylon can also be used. The separation pattern of the RNA molecules in the blotting paper remains the same as that in the gel.
- The blot is then exposed to a labeled, single-stranded DNA probe. The bases in this probe will pair with their complementary RNA sequences in the blot producing a double-stranded DNA-RNA molecule. Although the probe cannot be seen at this stage, since it is labeled with an enzyme or radioactive tag, it can be seen after appropriate treatment in the next step.
- Next, the probe is exposed to a colorless substrate which gets converted by the enzyme to a colored product and is visible on an X-ray film. In case the probe is radioactive, it can directly be seen on the X-ray film.
Applications
Some of the key applications of northern blotting are listed below:
- Gene expression studies – to observe overexpression of cancer-causing genes and gene expression in case of transplant rejection
- In diagnosis of several diseases, e.g., Crohn’s disease
- For detection of viral microRNAs that play key roles in viral infection
- To screen recombinants - by detecting the mRNA formed by the transgene
Limitations of northern blot
A few limitations of the northern blot technique are discussed below:
- The standard northern blot method has lower sensitivity when compared to that of RT-PCR and nuclease protection assays.
- The technique requires a large amount of target RNA sample sequence while newer techniques like real time RT-PCR need only a single copy of RNA for amplification.
- The northern blot technique is very sensitive to even slight degradation of the RNA samples. Degradation drastically affects data quality and quantitation.
- The technique becomes laborious in cases where multiple probes need to be added. A second probe can be added to the system only after removing the first probe completely. This can make the process highly complex. After treating the system with harsh chemicals to remove the first probe, the accuracy of the data from the subsequent rounds may also be compromised.
- Northern blot can only measure steady state mRNA accumulation and not RNA stability or transcription rates.
Summary
The northern blot technique is a classical way of analyzing the presence of a specific RNA sequence in a sample. Northern blotting is relatively cheap and simple to perform in the lab. Recent advancements in buffers and hybridization membranes have enabled high sensitivity blotting. However, breakthroughs in PCR technology in recent times have enabled a much more simple, quick, and precise identification as well as quantitation of RNA.
References
Further Reading
A step forward in vaccine technology: Exploring the effects of N1-methylpseudouridine in mRNA translation