Northern Blotting Procedure Steps

In this northern blotting procedure steps post we have briefly explained about northern blotting, principle, components, northern blotting technique procedure, applications, advantages and limitations.

Northern blotting

Northern blotting technique is based on the principle of blotting for the analysis of specific RNA in a complex mixture. The trend set by Southern blotting (in 1975) to detect specific DNA brought new ideas in the field of modern molecular biology.

When James Alwin, David Kemp, and George Stark of Stanford University recreated the design of the southern blot in 1977, they came up with something extremely similar to the southern blot. The most significant distinction was the utilisation of an RNA sample to identify a single RNA molecule within that sample.

Northern blotting technique for RNA detection was created by transferring cellular RNA to chemically activated cellulose paper. However, a radio-labelled DNA probe was still used in the procedure.

Definition

Northern blotting technique  used in molecular biology research to detect RNA or extracted mRNA in a sample and analyse gene expression (RNA blotting). Northern blotting technique is a common method for determining the size and steady-state level of a specific RNA in a complex sample.

It’s simple, inexpensive, and there are no artefacts in the way. Northern blotting technique, on the other hand, can present a number of technical challenges due to the process of RNA gel fractionation and probe creation, as well as variations in the quality of the blotting membrane used.

Principle

The basic idea behind northern blotting technique is to separate RNA by size using gel electrophoresis and then identify it on a cellular membrane using a hybridization probe containing a base sequence that corresponds to all or part of the target RNA chain.

To begin, we extract RNA from a tissue with chaotropic agents like guanidinium isothiocynate, which disrupts cells and denaturates proteins while also dissolving the RNA.

mRNA must be isolated from total RNA in some cases using a poly-A+ selection procedure. The resulting RNA is separated by agarose-gel electrophoresis and blotted onto a nylon membrane.

To prevent nucleic acid from washing away later, the RNA on the membrane must be immobilised by baking or exposure to UV light after blotting.

Finally, the hybridization probe is constructed and hybridised with the membrane. A post-hybridization wash is required to determine whether or not the probe is bound to the target mRNA. The signals are then detected and visualised using X-films and other methods.

Requirements

Equipments

  1. Agarose Gel cast
  2. Power Supply
  3. Microwave
  4. Centrifuge
  5. Heating block
  6. UV cross-linker
  7. Hybridization oven
  8. Hybridization vessels
  9. Vials
  10. Forceps
  11. Pipettes
  12. Glass tubes

Chemicals

  1. Agarose gel
  2. Bovine Serum Albumin
  3. Bromophenol Blue
  4. DTT
  5. Ethidium bromide
  6. Ethylenediaminetetraacetic acid disodium salt dehydrate
  7. Formaldehyde
  8. Glycerol
  9. HCl
  10. MgCl2
  11. NaCl
  12. NaH2PO4
  13. NaOH
  14. Polyvinylpyrrolidone
  15. RNA ladder
  16. SDS
  17. Sodium citrate
  18. Taq buffer
  19. Taq polymerase
  20. Tris-HCl
  21. Triton-X

Northern Blotting Procedure Steps

Northern blotting

Northern Blotting Procedure Steps

1. RNA Isolation

There are several methods for isolating RNA, but they all share some characteristics, such as cellular lysis and membrane disruption, inhibition of ribonuclease activity, deproteinization, and recovery of intact RNA.

RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction is one of the most common methods of RNA extraction.

RNA isolation using column technology. Spectrophotometry is used to assess the quantity and quality of RNA.

2. Electrophoresis

Formaldehyde is added to the agarose solution to make the RNA gel solution. The cast is put together, and the prepared denaturing gel is poured into it. As the gel begins to harden, a comb with the appropriate teeth is used to create wells.

The comb is removed after the gel has been set, and the gel is equilibrated with a running buffer for 30 minutes before running.

An equal volume of RNA loading buffer is mixed with 15 µg of RNA sample. In the same volume of RNA loading buffer, three g of RNA markers are added.

For about 12-15 minutes, the samples are incubated at 65°C on a heating block. The samples are loaded into the equilibrated gel, and the first row of wells is populated with RNA markers. After that, the gel is run at 125V for about 3 hours.

3. Transfer of RNA to a membrane

A nylon membrane larger than the size of the denaturing gel is prepared, as is filter paper the same size as the nylon membrane. After the electrophoresis, the RNA gel is removed from the tank and rinsed with water.

An oblong sponge slightly larger than the gel is placed on a glass dish, and the dish is filled with SSC to the point where the soaked sponge is half-submerged in the buffer.

A few pieces of Whatman 3mm paper are wetted with SSC buffer and placed on top of the sponge. The gel is then placed on top of the filter paper and squeezed out with a glass pipette to remove air bubbles.

On an RNase-free dish, the prepared nylon membrane is wetted with distilled water for about 5 minutes. The wetted membrane is placed on the surface of the gel while avoiding the formation of air bubbles.

SSC is applied to the surface once more, and a few more filter papers are placed on top of the membrane. To keep everything in place, a glass plate is placed on top of the structure. To ensure an effective transfer, the structure is left overnight.

4. Immobilization

When the transfer is finished, the gel is removed and rinsed with SSC before being allowed to dry. The membrane is sandwiched between two pieces of filter paper and baked for two hours in a vacuum oven at 80°C.

In some cases, the membrane can be wrapped in UV transparent plastic wrap and irradiated on a UV transilluminator for an appropriate period of time.

5. Hybridization

Hybridization with a radio or fluorescently labelled probe is used to identify specific RNA immobilisation. Single-stranded probes are prevented from binding to non-specific membrane sites by pre-hybridization.

The hybridization solution should contain 50% formamide to ensure hybridization at lower temperatures and minimise RNA degradation. To remove excess probes, the membrane is washed in a buffer containing less salt.

6. Visualization

Detection of specific transcript through autoradiography. Membranes are place over X-ray film. The X-ray film darkens where fragments are corresponding to the radioactive probes.

Applications

1. Northern blotting technique can be used to identify and separate RNA fragments obtained from various biological sources.

2. Northern blotting technique is a sensitive test for detecting the transcription of DNA fragments that will be used as a probe in Southern blotting.

3. Northern blotting technique also enables the detection and quantification of specific mRNAs from a variety of tissues and living organisms.

4. Northern blotting technique used to study gene expression in relation to cancer-causing gene overexpression and gene expression in transplant rejects.

5. Northern blotting technique has been used as a molecular diagnostic tool for diseases such as Crohn’s disease.

6. Northern blotting technique process is used as a method for the detection of viral microRNAs that play important roles in viral infection.

Limitations

1. Northern blotting technique is less sensitive than other modern techniques such as RT- PCR and nuclease protection assays.

2. Northern blotting technique method necessitates a large amount of sample RNA, which must be of high quality. The technique is time-consuming and complicated, especially when multiple probes are used.

Further Readings

Reference

  1. https://microbenotes.com/northern-blot/
  2. https://commons.wikimedia.org/wiki/File:Northern_blot_diagram.png
  3. https://www.thesciencenotes.com/northern-blotting-principle-procedure-and-applications/
  4. https://scigine.com/blog/rna-extraction-isolation-purification/
  5. https://experiments.springernature.com/articles/10.1007/978-1-4899-7708-3_12
  6. https://www.labmanager.com/insights/southern-vs-northern-vs-western-blotting-techniques-854