Microbiology

1. Northern Blotting
Presented TO:
Dr. Dildar Hussain Kalhoro
Associate Professor
Department OF Veterinary Microbiology
Presented & Prepared By
Hamid UR Rahman
2K16-VM-46

2. What is Blot?
In molecular biology blots is a technique for transferring DNA
, RNA and proteins onto a carrier so they can be separated,
and often follows the use of gel electrophoresis.
Types OF Bolting:
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

3. Northern Blotting

4. 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.

5. Northern Blotting

6. Applications
 Northern blots are particularly useful for determining the conditions
under which specific genes are being expressed, including which tissues
in a complex organism express which of its genes at the mRNA level.
 When trying to learn about the function of a certain protein, it is
sometimes useful to purify mRNA from many different tissues or cell
types and then prepare a northern blot of those mRNAs, using a cDNA
clone of the protein of interest as the probe.
 Only mRNA from the cell types that are synthesizing the protein will
hybridize to the probe.

7. Procedure
STEP 1 : ISOLATAION OF RNA :
All the target RNA’s molecules should be extracted from the
sample.
Isolate RNA from cells or tissue samples using the TRI
reagent or genelute™ kit for mammalian cells or tissues.
Step 2 : Agarose Gel Electrophoresis :
In gel electrophoresis the mixture of mRNA molecules
separated/denatured to small fragments/molecules according to their size
using an electric field.

8. Agarose Gel Electrophoresis
The sample should be pushed to electrical field
through a gel that containing small pores at a
speed that are inversely proportional to the size.
As we know that DNA/RNA have negative
charge due to the sugar-phosphate backbone
will move toward positive end of the field.

9. Agarose Gel Electrophoresis
Agarose & Buffer : Agarose & buffer are used is a gel to conduct
electrical current.
Formaldehyde :
Formaldehyde is used to unfragment the branched RNA molecule to
simple linear one and to prevent it form coiling again.

11. Gel Electrophoresis

12. Blotting/ Transfer
The transfer or blotting is the step in
which the mRNA from the
electrophoresis gel will be transferred
onto a nylon membrane so it may be
accessible to a probe for hybridization
and detection. The separated mRNA
bands are then blotted on chemically
reactive filter paper.

13. Blotting/ Transfer
Traditionally, a nitrocellulose membrane is used, although
nylon or a positively charged nylon membrane may be used.
Nitrocellulose typically has a binding capacity of about
100µg/cm, while nylon has a binding capacity of about 500
µg/cm. Many scientists feel nylon is better since it binds more
and is less fragile.
RNA,s will be covalently link to membrane.

14. Blotting/ Transfer

15. Hybridization/ Probe
In molecular biology hybridization means the process of
forming a double stranded nucleic acid from joining two
complementary strands of DNA (or RNA).
Pre-hybridization before hybridization blocks non-specific
sites to prevent the single-stranded probe from binding just
anywhere on the membrane.
Incubate membrane with labeled DNA or RNA probe with
target sequence.

16. Hybridization/ Probe
Incubate for several hours at suitable renaturation
temperature that will permit probe to anneal to its
target sequence(s).
Probe sequence is complementary to the RNA of
interest.

17. Hybridization/ Probe

18. Washing And Visualization
Wash Nylon from excess of probe & dry.
Place Nylon sheet over x-ray film.
X-ray film darkens where the fragments are complementary to
the radioactive probes.

20. Advantages/Application
 A standard for direct study of the gene expression at the level of
mRNA.
 Detection of mRNA transcript size
DISADVANTAGE :
 Time consuming procedure .
 Use of radioactive probes.
 Detection with multiple probes is a problem.

21. Northern And Southern Blotting Difference
Bothe are same in protocol but some difference as below:
Character Southern Northern
Introduction Southern blotting was
developed for the very
first time by the E.M.
Southern in 1975.
This method was
developed by the
and his colleagues in
1979.
Function For DNA For RNA
Denaturation Need of Denaturation No Need
Hybridization DNA-DNA hybridization RNA-DNA hybridization
Name Southern name of
Inventor
Northern is a misnomer