Northern blot technique Of RNA - Principle , Steps | Biology Blog

                                          Northern blotting 

Hello guys! In this blog, we are going to explore the northern blot technique. This technique can be used in any viral detection test as the genetic material of the virus is RNA.

So, before moving to northern blot we focus on the word - blot.

Blot is basically a technique in molecular biology and genetics in which we DNA/RNA or proteins could be transferred onto the carrier membrane. The nylon membrane or nitrocellulose membrane are used for blotting purpose. 

The transferring of molecules like DNA/ RNA or proteins to the membrane can take place after gel electrophoresis. After blotting the transferred molecules are visualized. 

For visualization, we can use the following methods- 

1. If the probe is labeled with the radioactive chemical (32P), it can be observed on X-ray film directly. 

2. If a chromogenic detection is used, it can be visualized by the development of color on the membrane itself. 

3. For the detection of DNA/RNA, a labeled probe is used. The labeling can be done with the radioactive isotopes (32P) or fluorescent dyes. 

So this is the blot technique, now we concentrate on the main topic - what is Northern blot and what is its steps.

Northern blot technique -  so guys, this technique was developed by Alwine and coworkers in 1977 and modified by  Thomas in 1980. In this technique, we can detect the presence of particular RNA in tissue samples.

Steps - it has the following steps - 

1. Isolation and purification - The RNA of interest is isolated by a high-speed centrifugation method and then it needs to purify.

2. Denaturation and electrophoresis - Denaturation is done for removing the fold in RNA and for this formaldehyde are used. After denaturation the shape of RNA becomes linear. Now, RNA is mixed with buffer solution and formaldehyde and run through gel electrophoresis. The electrophoresis separate RNA fragments. 

3. Blotting - After electrophoresis, blotting us done for transferring RNA into the nylon membrane. 

4.Incubation with the labeled probe - The membrane containing RNA is now incubated with a labeled probe. The probe used should be complementary to the RNA used. The probe used may be labeled with a radioactive chemical, not a fluorescent dye.

5. Hybridization- The membrane blot is soaked in polyethylene glycol and then washed in solution to remove unbound or loosely bound probe. 

6. Detection-  The labeled can be visualized by autoradiography. 

 Have you read - Western blotting techniques