Northern blotting

Briefly described about Northern blotting , Principle, their types and advantages of Northern Blotting

1. NORTHERN BLOTTING TECHNIQUE Prepared By ... Nabajit Roy M.Sc 2 nd Semester Paper –LS-202 Dept. of Life Science and Bioinformatics AUS 2017-2018

2. CONTENTS What is blotting Types of blotting Principle Northern blotting Application Advantage & Disadvantage Summary References 11-Apr-20 Northern blotting technique 2

3. What is Blotting? In molecular biology blotting is a technique for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follow the use of gel electrophoresis . 11-Apr-20 Northern blotting technique 3

4. Types OF Blotting: Types of blotting techniques are differentiated by the presence of the target molecules that is being sought. The first of this techniques developed was Dr. Edwin Southern (1975). Southern Blotting: Used For DNA Detection in a sample Northern Blotting : Used For RNA Detection in a sample Western Blotting : Used For Proteins in a sample Dot blotting technique: to detect the presence of a given sequence of DNA/RNA 11-Apr-20 Northern blotting technique 4

5. PRINCIPLE RNA are separated by size and detected on a membrane using a hybridization probe with a base sequence complementary to all, or a part, of the sequence of the target mRNA transfer of RNA from a gel to a membrane. And then the membrane is queried with a probe directed against the specific molecule of interest. 11-Apr-20 Northern blotting technique 5

6. NORTHERN BLOTTING The N orthern blot is a technique used in molecular biology to study gene expression by detection of RNA (or isolated mRNA) in a sample developed by Alwnie and his colleagues in 1979. This method was named for its similarity to the technique known as a Southern blot. 11-Apr-20 Northern blotting technique 6

7. 11-Apr-20 Northern blotting technique 7

8. APPLICATION Detecting a specific mRNA in a sample Used in the screening of recombinants by detecting the mRNA produced by the transgene. In disease diagnosis. In gene expression studies. 11-Apr-20 Northern blotting technique 8

9. ADVANTAGE The strength of this technique is its simplicity. Specificity is relatively high. mRNA transcript size can be detected. The cost of running many Gels is low once the equipment is setup. Blots can be stored for several years and reprobed if necessary. Quantity and Quality of RNA can be easily verified after electrophoresis and before further processing is done. DISADVANTAGE Risk of mRNA degradation during electrophoresis: Quality & Quantification of expression are negatively effected. High doses of radioactivity and formaldehyde are a risk for workers and the environment. The sensitivity of Northern blotting is relatively low in comparison with that of RT-PCR. Detection with multiple probe is difficult. Use of Ethidium Bromide ( EtBr ), DEPC & UV light needs special training and attention 11-Apr-20 Northern blotting technique 9

10. SUMMARY Northern blotting is the main method for examining the expression of genes through measurement of an mRNA . Or It is theoretically a good technique for determining the no. of genes (through mRNA) present on a given DNA. Tissue specificity and the factors which regulate expression can both be determined by Northern blotting. A cDNA radioactively labelled with 32P is the most-commonly-used hybridization probe. Convenient, non-radioactive detection protocols are, however, increasingly available. A combination of anti-sense oligonucleotides as probes, together with chemo-luminescence-based detection provides a rapid and simplified approach to Northern blotting, increasing the accessibility of this important procedure for nutritional studies. 11-Apr-20 Northern blotting technique 10

11. REFERENCES Wilson, K & Walker, J ,(2010), Principles and techniques of biochemistry and molecular biology. Satyanarayan , U.(2015) biotechnology pp. 68-101 Goda , S.K. & Nigel, P.M., “A simple procedure for gel electrophoresis and northern blotting of RNA”(1995) “Nucleic acid research 23.16:3357 Trayhurn , P.(1996). Northern Blotting Proceedings of the Nutrition society. Pp 55, 583-589. 11-Apr-20 Northern blotting technique 11

12. 11-Apr-20 Northern blotting technique 12