Northern blotting presentation

1. Northern blotting
BY-SANJU SAH
ST.XAVIER’S COLLEGE, MAITIGHAR,
KATHMANDU
DEPARTMENT OF MICROBIOLOGY

2. Introduction
 The northern blot, or RNA blot, is a technique used
in molecular biology research to study gene
expression by detection of RNA (or isolated mRNA)
in a sample
 Detect specific RNA molecules among a mixture of
RNA.
 Used to analyze a sample of RNA from a particular
tissue or cell type in order to measure the RNA
expression of particular genes.
 Technique developed in 1977 by J.Alwine, D. Kemp &
G. Start .

3.  mRNA is generally represented as 5% of the
overall RNA sequence.
 This method reveals the identity, number,
activity, and size of the particular gene.
 It also aids in the identification of abnormal,
diseased or infected condition at the molecular
level.
 Observe cellular control over structure and
function by determining the particular gene
expression rates during differentiation
and morphogenesis.

4. Principle
 Northern blotting involves the use
of electrophoresis to separate RNA samples
by size, and detection with a hybridization
probe complementary to part of or the
entire target sequence.
Capillary transfer of RNA from the
electrophoresis gel to the blotting
membrane.

5. Steps involved
 Extract and purify mRNA from cell
 Seperation by Gel electrophoresis
 Blotting
 Hybridization
 Wash off unbound probe
 Detection by autoradiograph

7. Procedure
1. Extraction :Eukaryotic mRNA can be isolated
through the use of oligo (dT)
cellulose chromatography[ to isolate only those
RNAs with a poly(A) tail
2. Separated by gel electrophoresis : The gels are
fragile and the probes are unable to enter the
matrix and separated by size
3. Blotting : Transferred to a aminobenzyloxymethyl
filter paper through a capillary or vacuum as it
lowers the annealing temperature of the probe-
RNA interaction, thus eliminating the need for high
temperatures, which could cause RNA degradation

8. 4.After transfer the membrane is baked at 80⁰ C for
fixation.
5. The membrane is then put into a solution containing
probes so that the probe will Hybridize only on a
specific Target mRNA to form RNA-DNA duplex .
6. The membrane is washed to ensure that the probe
has bound specifically and to prevent background
signals from arising.
7. Labeled probe is detected by chemiluminescence or
autoradiography. The result will be dark bands in x ray
film.

9. Applications
• Detection of specific mRNA in a sample
• Used in screening of recombinants by
detecting the mRNA produced by the
transgene
• Disease diagnosis
• Gene expression study as in case of
oncogenes and transplanted organ

10. Advantage
 Simple method.
 Highly specific.
 Quality and Quality of gene can be measured.
 Detection of RNA size
 Observation of alternate splice products
 The quality and quantity of RNA can be
measured on the gel prior to blotting, and
 The membranes can be stored and reprobed
for years after blotting.

11. Disadvantage
 Time consuming.
 Detection with multiple probe is problem.
 Use of radioactive probed.
 Use of UV light needs special training.
 Detect small number of gene at once.