Northern Blot

Northern Blot. It is a laboratory based technique for identification of RNA (Ribonucleic acid) molecules. This technique was first developed by James Alwine, David Kemp, and George stark. This Northern Blot or Northern Hybridization technique is very similar to the Southern blot or Southern hybridization technique except that in the northern blot technique RNA is identified instead of DNA.  

Northern Blot / Northern Hybridization

Definition

Northern Hybridization is the laboratory technique that is used to detect specific RNA (Ribonucleic acid) molecules among a mixture of RNA samples. It is also called northern blotting.

It is a technique with the help of which we can study the gene expression in a cell by detection of RNA or isolated mRNA in a sample

It also helps in understanding the overexpression of the oncogenes when compared with the expression of genes in normal tissue

Principle 

Northern Hybridization principle is based on the separation of mRNA based on their size and charges via gel electrophoresis which is afterward transferred to the membrane and is then detected by hybridizing them with their complementary probes

Terms that ought to be understood first:-

Hybridization= When the single-stranded RNA or DNA molecule (probe) that pairs with another complementary single-stranded RNA or DNA (target) molecule is termed as hybridization. In different words, it is the pairing of two single-stranded oligonucleotides together

Probes = It is a short single stranded oligonucleotide of RNA or DNA molecules which are complementary to the target sequences. These are produced artificially. They are tagged with different radioactive elements (32P) or with non radioactive elements (fluorescently tagged)

Blotting= Blotting is the transfer of DNA, RNA, or protein from gel to a membrane (nitrocellulose or nylon membrane) with the help of capillary action or electric field.

Procedure

1. Isolation of RNA

2. Separation of RNA using gel Electrophoresis

You see, that the gel we prepare is agarose gel. When this agarose gel solidifies it becomes gelly like or gel like appearance we can see. If we observe this solidified mass of gel under the microscope we see lots of pores in it. So we prepare our samples of RNA and when we load them into the wells of the agarose, this gel act as a sieve and filters out our RNA molecules based on their size and length and charge on that nucleic acid molecule. So when we apply current to the gel the small molecules of RNA move fast towards the anode through the gel's small pores cause the molecule size is small. Whereas the large RNA molecules can travel a small distance because their size is large and so they seem to be stuck in the pores of the gel.

Preparation of gel for northern blot-

     Prepare an agarose gel and before loading our isolated RNA samples within the well, first denature the RNA molecules with the use of formaldehyde or DMSO(dimethyl sulfoxide) because RNA is single-stranded molecule so it has a tendency to pair with other RNA molecules or pair with itself; because of which RNA molecules do not run efficiently through the gel, so by adding formaldehyde or DMSO(dimethyl sulfoxide) it disrupts the secondary structure of the RNA and makes it linearized. Next run the gel, and the RNA samples will separate on the basis of their size and charge.

3. Transfer of RNA to a membrane

    It is the blotting step, in here we use a nylon membrane. It can be carried out in two ways via capillary method or electrophoretic transfer method. After the gel is run the gel is kept on the filter paper and on top of the gel the nylon membrane is kept and after nylon more filter paper is added; all this apparatus is kept in a vessel; in case of capillary transfer where a transfer is carried out with the capillary mechanism or in an electrophoretic apparatus where the transfer is carried out with the help of electric field. Electric impulse is applied to the nucleic acid that is attached to gel cathod(-) gets transferred to the nylon membrane which is placed towards the anode(+). After the blotting step is done the nylon membrane is taken out and baked at around 80 oC OR UV (ultraviolet rays) crosslinked to immobilize our RNA on the membrane.

4. Hybridization and Washing

    The membrane is taken out and is flooded with various types of buffers like pre-hybridizing buffer, and hybridizing buffers, here probes are added and then they are washed with the washing buffer.

1) Pre-hybridizing buffer = Pre-hybridizing buffer goes and block those site on the membrane where no RNA is bound ensuring that when we perform hybridization our probes get attached to only our RNAs and not the membrane 

2) Hybridization buffers= Hybridization buffer help in the hybridization of our probe with the target RNA. 

3) Washing buffer = Washing buffers ensure that after the hybridization step no access unbound probe remained on the membrane. 

5. Visualization

     If used radioactive tags, RNA bands are observed in the autoradiography or if used fluorescent tag we can observe bands with the naked eye.

Must read: Protoplast Culture: Isolation

Advantages of Northern Hybridization

1) With the help of northern hybridization, we can detect a particular RNA of interest in a mixture of RNA samples.

2) It also helps us to understand the size of an RNA molecule by comparing it with the corresponding standard ladder. 

3) We can study the gene expression rate in a cell.

4) It also helps us in the detection of various diseases.

5) Since we can isolate RNA and study we can compare an agarose slab of a normal gene and an agarose slab of a cancerous gene and understand the overexpression of a particular gene by comparison study.

6) We can also study about many viral infections since most of the viruses have RNA as genetic material.

7) This technique helps in the detection of many viral infections.

Conclusion

Here we understood the application of Northern Blot, why we perform it along with its definition, along with all the steps in northern blot. This technique is done for RNA isolation where it is possible for us to detect our RNA of interest in a mixture of RNA samples, whereas southern blot is done for DNA samples in there it helps us in the criminal investigations, parental child disputes, DNA fingerprinting, and many other things.We even perform western blot / western hybridization in here it used for the identification of specific proteins in a mixture of proteins in a sample. 

Must read: Probiotics and Prebiotics