1. Name : Kainat Sultana
Roll no : 17411506-051
Submitted to : Sir Naeem Abbas
Semester : 7th
Topic : Northern Blothing
University of chakwal
2. Northern blothing
A northern blot is a laboratory method used to
detect specific RNA molecules among a mixture
of RNA.
Northern blotting can be used to analyze a
sample of RNA from a particular tissue or cell
type in order to measure the RNA expression of
particular genes.
This method was named for its similarity to the
technique known as a Southern blot.
.
3. The northern blot technique was developed in
1977 by James Alwine, David Kemp, and
George Stark at Stanford University, with
contributions from Gerhard Heinrich. Northern
blotting takes its name from its similarity to the
first blotting technique, the Southern blot, named
for biologist Edwin Southern.
The major difference is that RNA, rather than
DNA, is analyzed in the northern blot.
5. Northern blothing is a method used to study gene
expression by detection of RNA in a simple.
Therefore, it is also called RNA blot.
the simple RNA is isolated fron an organismof
interest and then electrophoresed on agarose gel
which separates the fragments on the basis of
their size.
The separated RNA fragments are transffered to a
support membrane(nitrocellulose membrane).
This can be performed by simple capillary method
in prescence of a specific buffer.
6. The RNA is then immobilized on membrane either
baking at high temperature or UV crooslinking,
which results in covalent linkage of RNA to
membrane preventing nucleic acid from being
washed away from subsequent processing.
FIG 2
7. This step is followed by hybridisation with a
labeled DNA or RNA probe. If the sample contains
the complementary RNA sequence, the probe will
bind to membrane to form doubled 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.
8. Agarose gel for process of gel electrophoresis.
Nylon membrane / diazo benzyl oxy methyl filter
paper.
Complementary radioactive probe for
hybridization.
Formaldehyde(HCHO) for degradation.
X-ray film for idendification of RNA.
9. RNA isolation
Separation of RNA using gel electrophoresis
Blothing
Hybridization and washing of probes
Visualization
10. RNA gel electrophoresis
The tissue or culture sample collected is first
homogenized. The samples may be
representative of different types of culture for
comparison or it can be for the study of different
stages of growth inside the culture.
The RNA sequence is separated in the
electrophoresis unit an agarose gel is used for the
purpose of the nucleic acid separation.
11. Now the separated RNA sequence is transferred
to the nylon membrane. This is done by two
mechanisms capillary action and the ionic
interaction.
The transfer operation is done by keeping the gel
in the following order. First, the agarose gel is
placed on the bottom of the stack, followed by the
blotting membrane. On top of these paper towels
a mild weight (glass plate) is placed. The entire
setup is kept in a beaker containing transfer buffer.
12. RNA transferred to the nylon membrane is then
fixed using UV radiation.
The fixed nylon membrane is then mixed with
probes. The probes are specifically designed for
the gene of interest, so that they will hybridize with
RNA sequences on the blot corresponding to the
sequence of interest.
The blot membrane is washed to remove
unwanted probe.
13. Labeled probe is detected by chemiluminescence
or autoradiography.
The result will be dark bands in x ray film.
14. The RNA samples are separated using agarose
gels using formaldehyde as denaturing agents but
in small RNA or micro RNA sequences,
polyacrylamide sequences with urea as a
denaturing agent also can be used.
Ethidium bromide can be used as a staining
agent. Two types of markers are for size marking.
An RNA ladder and ribosomal subunit are used for
the identification of the size of the RNA
sequences.
15. Probes can be complementary to the whole or part
of the RNA of interest.
They can be RNA, DNA or oligonucleotides of 25
complementary basepairs to the target RNA.
In case of RNA probes, invitro produced probes
are used as invivo probes can denature due to the
rigorous washing.
In case of cDNA, the probes are labeled with
radioactive isotopes, alkaline phosphatase or
horseradish peroxidase in case of
chemiluminescence.
17. The advantages of using northern blotting include
the detection of RNA size, the observation of
alternate splice products, the use of probes with
partial homology, 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.
Blots can be stored for several years and reported
if necessary.
18. RNA splicing is visible because alternatively spliced
transcripts can be detected.
It is also useful in detection of mRNA transcript size.
Specifity is relatively high.
19. A problem in northern blotting is often sample
degradation by RNases (both endogenous to the
sample and through environmental
contamination), which can be avoided by proper
sterilization of glassware and the use of RNase
inhibitors such as DEPC (diethylpyrocarbonate).
The chemicals used in most northern blots can be
a risk to the researcher, since formaldehyde,
radioactive material, ethidium bromide, DEPC,
and UV light are all harmful under certain
exposures.
20. Compared to RT-PCR, northern blotting has a low
sensitivity, but it also has a high specificity, which
is important to reduce false positive results.
