A person with a genetic mutation expresses a new or foreign protein that may or may not be harmful. Many techniques help in determining the type of protein and the reason for its expression. One of them is northern blotting. Like all the blotting techniques, northern blotting is the process of blotting separated RNA in a membrane, hybridizing it with the probe, and quantifying the RNA. In short, Northern blotting is the process of measuring or quantifying RNA, especially mRNA.
Objective of Northern Blotting
The need to measure the RNA emerged to:
- Identify the types of protein secreted by specific tissues.
- And also determine what the regulating factors of the gene expression are.
Principle of Northern Blotting
- The principle of the method is the separation of RNA according to size by agarose gel electrophoresis and afterwards transferring the separated RNA on a nylon membrane.
- Consequently, the detection of the desired RNA is done by using the radiolabeled probe with all or a part of the base sequence of the targeted RNA hybridizing with the immobilized and separated RNA
Materials and Equipment Required
- 20X MAE
- Gel electrophoresis equipment
- 20X SSPE
- RNA loading buffer
- Loading dye (10X)
- Nylon hybridization membrane (3MM Whatman paper)
- Capillary system
- Ultraviolet light transilluminator
- NTPs: UTP, ATP, GTP, CTP
- Micropipette tips
- Centrifuge tubes
- DNA template
Procedure of Northern Blotting
RNA Extraction and Separation
- Firstly, prepare an agarose gel and pour it into a casting tray.
- Then let the gel solidify.
- After that, run the running buffer for equilibration for about 30 minutes.
- Simultaneously, mix the sample with RNA loading buffer.
- Then, add RNA marker to the mixture above and incubate.
- After that, mix the RNA sample mixture in the equilibrated gel.
- At last, run the gel at 125V for approximately 3 hours.
Transfer of the Separated RNA
- At first, cut a nylon membrane and 3 MM Whatman paper in the same size as denaturation gel.
- Afterwards, place the gel above a sponge for support in a vacuum container containing SSC (transfer buffer).
- Then, position the agarose gel in such a way that the separated RNA faces above.
- After that, place the cut and wet nylon membrane on top of the gel.
- Similarly, place the 3 MM Whatman paper above the membrane.
- Then, vacuum pressure is provided in the container.
- The estimated time for the transfer is 90 minutes.
Fixing the Separated RNA in the Membrane
- After the transfer completes, cross-link with UV to fix the RNA in the membrane first.
- Then, repeat the Crosslinking process.
Radio labeling the Probe
- Firstly, prepare a probe similar to the target RNA with NTPs.
- Then, radiolabel the probe.
- Finally, introduce the probe to the membrane.
Hybridization and Analysis
- Firstly, hybridize the probe by placing the membrane in hybridizing solution.
- Then, wash the membrane after hybridization
- At last, analyze the membrane under X-ray and use software for quantification.
Things to Remember
The precautionary measure necessary for performing this method are:
- Maintain RNase free environment to keep RNA intact.
- Follow personal safety measures.
- Cool down the gel before adding formaldehyde.
- Handle and dispose of radioactive labeling probes according to the protocol.
Advantages of Northern Blotting
- It helps in the detection of small changes in RNA.
- It has high specificity.
- It is also a quantitative method.
Disadvantages of Northern Blotting
- It has less sensitivity.
- Also, maintaining an RNases-free medium is challenging.
- Likewise, the analysis of a large number of genes becomes tedious.
References and Further Reading
- He, S., & Green, R. (2013). Northern Blotting. Laboratory Methods In Enzymology: RNA, 75-87. https://doi.org/10.1016/b978-0-12-420037-1.00003-8
- Lovatt, D., & Eberwine, J. (2013). Northern Blotting. Brenner’s Encyclopedia Of Genetics, 105-107. https://doi.org/10.1016/b978-0-12-374984-0.01065-2
- Murphy, D. Northern Blotting. Transgenesis Techniques, 337-340. https://doi.org/10.1385/0-89603-245-0:337
- Trayhurn, P. (1996). Northern blotting. Proceedings Of The Nutrition Society, 55(1B), 583-589. https://doi.org/10.1079/pns19960051
- Northern Blotting Protocol, Principle, Application, Result. Microbiology Note. (2022). Retrieved 29 April 2022, from https://microbiologynote.com/northern-blotting-technique/.