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NR
Uploaded byNabajit Roy
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Northern blotting

Northern blotting is a technique used to detect specific mRNAs in a sample. It involves separating RNA fragments by size through gel electrophoresis, transferring them to a membrane, then using a complementary DNA or RNA probe to identify the mRNA of interest. It allows researchers to examine gene expression by measuring mRNA levels and determine tissue specificity and regulatory factors of gene expression. While it provides useful information, it also has some limitations such as risk of mRNA degradation and relatively low sensitivity compared to PCR-based methods.

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NORTHERN BLOTTING TECHNIQUE Prepared By... Nabajit Roy M.Sc 2nd Semester Paper –LS-202 Dept. of Life Science and Bioinformatics AUS 2017-2018
CONTENTS  What is blotting  Types of blotting  Principle  Northern blotting  Application  Advantage & Disadvantage  Summary  References 11-Apr-20 Northern blotting technique 2
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
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
PRINCIPLE 1. 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 2. transfer of RNA from a gel to a membrane. 3. And then the membrane is queried with a probe directed against the specific molecule of interest. 11-Apr-20 Northern blotting technique 5
NORTHERN BLOTTING  The Northern 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
11-Apr-20 Northern blotting technique 7
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
ADVANTAGE 1. The strength of this technique is its simplicity. 2. Specificity is relatively high. 3. mRNA transcript size can be detected. 4. The cost of running many Gels is low once the equipment is setup. 5. Blots can be stored for several years and reprobed if necessary. 6. Quantity and Quality of RNA can be easily verified after electrophoresis and before further processing is done. DISADVANTAGE 1. Risk of mRNA degradation during electrophoresis: Quality & Quantification of expression are negatively effected. 2. High doses of radioactivity and formaldehyde are a risk for workers and the environment. 3. The sensitivity of Northern blotting is relatively low in comparison with that of RT-PCR. 4. Detection with multiple probe is difficult. 5. Use of Ethidium Bromide (EtBr), DEPC & UV light needs special training and attention 11-Apr-20 Northern blotting technique 9
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
REFERENCES 1. Wilson, K & Walker, J,(2010), Principles and techniques of biochemistry and molecular biology. 2. Satyanarayan, U.(2015) biotechnology pp. 68-101 3. Goda, S.K. & Nigel, P.M., “A simple procedure for gel electrophoresis and northern blotting of RNA”(1995) “Nucleic acid research 23.16:3357 4. Trayhurn, P.(1996). Northern Blotting Proceedings of the Nutrition society. Pp 55, 583-589. 11-Apr-20 Northern blotting technique 11
11-Apr-20 Northern blotting technique 12