This presentation provides an overview of northern blotting. Northern blotting is a technique used to detect RNA in a sample. It involves separating RNA fragments via gel electrophoresis, transferring them to a membrane, then using a labeled probe to detect the RNA of interest. The key steps are isolating RNA from cells, separating fragments by size via gel electrophoresis, transferring RNA to a membrane, hybridizing the membrane with a labeled probe, and detecting the RNA of interest on the membrane. Northern blotting allows researchers to study gene expression patterns and RNA splicing.

1. Presentation on Northern Blotting
Prepared by Zeshan Zafar
Reg# L1F19MCBT0009

2.  1) Defination of blot
 2) types of Blot
 Northern Blotting
 Principle
 Procedure
 Application
 Advantages
 disadvantages

3. Northern Blotting
What is Blot?
Blot is a technique used to detect DNA RNA proteins onto a carrier
so they can be separated and often fallow gel electrophoresis.

4. Types of Blot
 There are fallowing three types of blotting which are
given below as fallow,
 Southern Blotting: which can be used to detect DNA in a
sample.
 Northern Blotting: which can be used to detect RNA in a
sample.
 Western Blotting: which can be used to detect proteins
in a sample.

5. Northern Blotting
 Northern Blotting is technique in molecular biology
research to study expression of gene by detection of
RNA.
 It is also known as RNA blot is one of the blotting
techniques used to transfer DNA and RNA onto a carrier
for sorting and identification.
 Northern blot is similar to southern blot except RNA
instead of DNA.

6. Northern Blot
 It is messenger RNA which is isolated and hybridized in
northern blot.

7. Discovery of Northern Blotting
 Northern Blsotting was developed in 1977 by james
Alwaine , David kemp and George stark at Stanford
univeristy.

8. Principle of Northern Blotting
 Is a method used to study gene expression by the
detection of RNA in a sample therefore it is also called
RNA blot.
 The sample RNA is isolated from an organism of interest
then electrophoresed on agarose gel which separates the
fragment on the base of their size.
 The separated RNA fragment are transferred to a
support membrane ( Nitrocellulose membrane).

9. Principle of Northern Blot
 The RNA is then immobilized on membrane either by
baking at high temperature or UV crosslinking which
result in covalent linkage of RNA to membrane
preventing nucleic acid from being washed away from
subsequent process.
 This is fallowed by labeled hyridization with a DNA or
RNA probe.

10. Principle of Northern Blot
 If the sample contain the complementary RNA sequence
the probe will bind to membrane to form double
stranded DNA-RNA hybrid molecule between single
stranded DNA probe and single stranded target RNA.
 The final step is the detection of RNA of interest on the
membrane using chromogen.

11. Procedure
 STEP 1:
 DNA containing the gene of interest is extracted from
human cells and cut into fragments by restriction
enzyme.
 STEP 2:
 The fragment are separated according to size by gel
electrophoresis. Each band consist of many copies of a
particular DNA fragment.

12. Procedure
 The DNA bands are transferred to a nitrocellulose filter
by bloting the solution passes through the gel and filter
to the paper towels.
 This produce a nitrocellulose filter with DNA fragments
position exactly as on the gel.
 STEP 5: The filter is exposed to radioactively labelled
probe for a specific gene.
 The probe will base pair with a short sequence present
on the gene.

13. Procedure
 The filter is then exposed to x ray film.
 The fragment containing the gene of interest is identified
by a band on the developed film.

14. Applications
 Observe a particular gene expression pattern b/w tissue
organ development stages environment stress etc
 Detecting a specific messenger RNA in sample used for
screening recombinant which are succefuly transformed
 Also used for messenger RNA suplicing.

15. Advantages
 Northern blot are particularly useful to determine
condition which specific gene are expressed.
 It is useful in detection of mRNA transcript size.
 RNA splicing is visible because alternative splicing
transcript can be detected.
 Blot can be stored for several years and reprobed if
necessory.

16. Disadvantage
 Risk of mRNA degradation during electrophoresis quality
and quantification are negatively effected.
 High doses of radioactivity and formaldehyde are a risk
for workers and environment.
 Use of ethidium bromide DEPC and UV light need special
training and attention.