It is most important ppt of northern blotting to educate the basic learner of Biotechnology

1. Northern Blotting
By:
Dr. Ashish Patel,
Ph.D.Scholar,
Animal Genetics & Breeding

2. • The northern blot is a technique used to study
gene expression by detection of RNA in a sample.
Or
• A northern blot is a method used to detect
specific RNA molecules among a mixture of RNA
and also be used to analyze a sample to measure
the RNA expression of particular genes.
• The name of northern blot method derived from
its similar first blotting technique known as a
Southern blot (which was invented by Edwin
Southern).
• Northern blotting was developed by Alwine in
1977.

3. Principles of Northern blotting
• Electrophoresis – Seperates RNA on the basis of
their Molecular weight and type in agarose gel
which have EtBr ,an intercalating agent in it.
• Capillary action – RNA bands move towards
blotting paper by capillary movement and entrap
in sheet and buffer moves ahead.

4. Steps involving Northern Blotting
• RNA isolation
• Separation of RNA using Gel Electrophoresis
• Blotting
• Hybridization and Washing of excess probes
• Visualization
• The Northern Blot procedure is quite similar
to that of Southern blot, except that the
biomaterial used is RNA instead of DNA.

5. 1. RNA ISOLATION:
• The RNA is isolated from the cell.
• All protocols, techniques, and commercially
available kits used to isolate RNA with
common attributes:
Cellular lysis and membrane disruption
Inhibition of ribonuclease activity
Deproteinization
Recovery of intact RNA

6. Methods of RNA extraction
1. RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform
extraction
Principle:
RNA is separated from DNA after
extraction with an acidic solution
containing guanidinium thiocyanate,
sodium acetate, phenol and
chloroform, followed by
centrifugation.
Under acidic conditions, total RNA
remains in the upper aqueous phase,
while most of DNA and proteins
remain in the lower organic phase.
Total RNA is then recovered by
precipitation with isopropanol and can
be used for several applications.

7. 2. RNA isolation by Column-based
Technology
Principle:
Nucleic acid will bind to the solid
phase of silica under certain
conditions.
A buffer solution along with sample,
ethanol or isopropanol forms the
binding solution and the binding
solution is transferred to a spin
column and the column is put in a
centrifuge. The centrifuge forces
the binding solution through a
silica gel membrane that is inside
the spin column and nucleic acid will
bind to the silica gel membrane

8. 3. RNA isolation by Chemicall cum
Column-based Technology

9. Quality assessment of total RNA
• RNA quality can be assess by Nano Drop
technology or by Bioanalyser
• There are three quality controls that are performed
on isolated RNA.
Via Nanodrop:
• Quantity of RNA: measure in ng/µl.
• Purity of RNA: The ratio of the absorbance at 260
and 280 nm is used to assess the RNA purity of an
RNA preparation. Pure RNA has an A260/A280 of
ranges 1.8 to 2.0.
• Integrity of the RNA: can be measured on
bioanalyzer

11. • Bioanalyzer: perform capillary electrophoresis and uses a
fluorescent dye that binds to RNA to determine both RNA
concentration and integrity.
• it is recommended to use at least 50 ng/μl for a
meaningful RNA Integrity Number (RIN).
• The RNA Integrity Number (RIN) software algorithm allows
the classification of total RNA, based on a numbering
system from 1 to 10, with 1 being the most degraded and
10 being the most intact.
• If RIN of 5 might not work for a microarray experiment,
but might work for an RT-PCR experiment.
• In general, RINs higher that 7-8 seem to be working well in
most of experiments.
• RINs below 7 require extra validation studies before we
are able to conclude “how bad is still good enough".

13. 2. SEPARATION OF RNA
• Once RNA samples are isolated, the next step is
denaturing agarose gel electrophoresis.
• Types of gel: Agarose (for DNA and RNA: source: sea
weed), Polyacrylamide gel (for Protein) and Starch (for
Protein).
• Gel Conditions:
• 1. Denaturing gels: Disrupt the natural structure of
DNA / RNA and causing it to unfold into a linear chain.
Thus, the mobility of each macromolecule depends
only on its linear length and its mass-to-charge ratio.
• Thus, the secondary, tertiary, and quaternary levels of
biomolecular structure are disrupted, leaving only the
primary structure to be analyzed.
• Nucleic acids denatured by urea/formaldehyde in the
buffer, while proteins are denatured using sodium
dodecyl sulfate.

14. • 2. Native gels: non-denaturing conditions, so that the
analyte's natural structure is maintained.
• In Northern blotting, Formaldehyde has been the
denaturant traditionally used during electrophoresis.
• Formaldehyde agarose gel electrophoresis is normally
used in the separation of RNA as formaldehyde agarose
gel prevent RNA from folding on itself.
• The disadvantage of using formaldehyde is the need to
pour and run gels in a fume hood.
• On electrophoresis RNA molecules moves towards
positive pole as RNA is negatively charged.

15. Choice of buffer for gel electrophoresis
• Factors for choosing a buffer:
• A simple buffered solution contains a mixture of a weak acid
(HA) and its conjugate (A-) base.
1. Formal charges of buffer species: - Generally, buffers which
form ions of high charge magnitude (+2, +3, -3, etc.).
Resulting in to at relatively low concentrations, the gel
conducts too much current. Furthermore, with ions moving
quickly through the gel, the buffer may become depleted.
One of the reasons Tris-borate is a popular buffer for
electrophoresis is that both Tris base and borate are
uncharged part of the time at the desired pH, which reduces
their electrophoretic mobility.
2. Molecular size - Low charge Tris base moves slowly in
electrophoresis because of its relatively large molecular size.
Having a low charge to mass ratio, Tris moves much more
slowly than small ions such as chloride or phosphate.

16. 3. pKa value (Acid dissociation constant) - A buffer should be
chosen with a pKa that is very close to the desired pH.
Other factors to consider when choosing a buffer would
include toxicity, solubility, UV absorption and the possibility
of interaction with other species present in the solution.
There are a number of different buffer configurations that
are used for different kinds of electrophoresis
Homogenous Buffer System - the identity and concentration of
buffer components are the same in the gel and both tanks.
This is used for most forms of DNA and RNA
electrophoresis.
Buffer Molecular Weight (Mr) pKa
Acetic Acid 60.05 4.8
Boric Acid 61.68 9.23
Glycine 75.07 9.8
Tricine 179.18 8.15
Tris 121.1 8.06

17. • Multiphasic Buffer Systems - System uses differing buffers
and is used for SDS-PAGE (often called the Laemmli system).
The Laemmli system uses an additional gel layer above the
separating gel.
Buffers used are:
Stacking Gel - Tris-HCl, pH 6.8, Separating Gel - Tris-HCl, pH
8.8 at a higher concentration, Tanks - Tris-glycine, pH 8.8.
• Buffer Additives - are usually added to the buffer perfusing
the gel and can include:
Hydrogen bonding agents: Urea and formamide, which
disrupt hydrogen bonds, that affect the conformation and
solubility of molecules.
Surfactants : Triton X-100, Tween 20 or SDS. SDS is the most
commonly used detergent. This causes the protein chains to
unwind from their native configuration and the protein is said
to be denatured.
Reducing Agents : 2-mercaptoethanol or dithiothreitol that
break the disulphide bond linkages that hold protein chains
together. The protein is then said to be reduced.

18. 3. BLOTTING
• Simply blotting is process of transferring the RNA molecules to
the nitrocellulose membrane or nylon membrane:
Choice of membrane:
• There are several types of commercially available membranes
suitable for RNA analysis, composed of different materials and
carrying different charges.
• The common ones are made of nylon and nitrocellulose, and
may be neutral, negatively or positively charged.
• Nylon (polyamide) membranes are made of the most durable
material, but can shrink or warp if exposed to organic solvents.
• Nitrocellulose tends to tear easily in washing steps and
becomes very fragile.
• Negative membranes give the cleanest background, but result
in poor specific signal.
• Positively charged membranes give the best signal of all, but
they also result in higher background.
• Many scientists feel nylon is better since it binds more and is
less fragile.

19. • Three types of blotting membranes are available:
Membra
ne
Properties Applications Pore sizeRepro
bing
Nitrocell
ulose
Most widely used membrane for western blotting
Good binding capacity
Proteins bind to the membrane due to hydrophobic
interactions
Protein binding capacity: 80 µg/cm2
Western transfer
Amino acid analysis
Solid phase assay
systems
0.2 µm
0.45 µm
No
PVDF Higher binding capacity than nitrocellulose
Strong hydrophobic character and solvent resistant
Physically stronger than nitrocellulose
Compatible with commonly used protein stains and
immunodetection methods
Protein binding capacity: 50-150 µg/cm2
Protein sequencing
Western transfer
Amino acid analysis
Solid phase assay
systems
Yes
Nylon Microporous membrane modified with strongly
basic charged groups
Ideal for binding negatively charged biomolecules
such as DNA and RNA
Low background for enhanced resolution
Membrane is formed around a non-woven polyester
fiber matrix which confers high tensile strength,
toughness, and flexibility
Southern and
northern transfers
Solid phase
immobilization
Dry chemistry test
strips
Enzyme
immobilization
Gene probe assays
0.45 µm Yes

20. • Once separated by denaturing agarose gel electrophoresis, the
RNA is transferred to a positively charged nylon membrane and
immobilized for subsequent hybridization.
• For fast, reproducible transfer, the iBlot Dry Blotting System
offers complete transfer of RNA to nylon membrane typically in
7 minutes. With dry blotting, there is no need for additional
buffer or liquids that can introduce variability into the result.
• This system is compatible with both polyvinylidene difluoride
(PVDF) and nitrocellulose membranes, and has comparable
performance to traditional wet transfer methods in a fraction
of the time.
• High detection sensitivity
• Increased blotting reliability and reproducibility
• Flexible gel-size formats and membrane types
• High-quality and more compact transfer stacks

21. iBlot Dry
Blotting System
Semi-dry
transfer
Wet or semi-
wet transfer
Buffer preparation 0 minutes 30 minutes 30 minutes
Soaking gel in transfer buffer 0 minutes 20 minutes 0 minutes
Assembling layers 2 minutes 10 minutes 10 minutes
Transfer 7 minutes 45–90 minutes 1–3 hr
Cleanup 0 minutes 10 minutes 10 minutes
Total elapsed time 9 minutes
1 hr, 55 min–
2 hr, 40 min
1 hr, 50 min–
3 hr, 50 min
Time saved with the
iBlot Dry Blotting System
—
1 hr, 45 min–
2 hr, 30 min
1 hr, 40 min–
3 hr, 40 min
Comparison of elapsed time for protein transfer with the iBlot Dry
Blotting System to other blotting methods.

22. iBlot Dry Blotting System Semi-dry transfer
Wet or semi-wet
transfer
Preassembled stacks ready for
protein transfer containing
electrodes, buffer matrices,
and PVDF or NC membrane
Transfer stack (both top
and bottom) composed of
sponge and filter paper,
soaked in buffer
Transfer stack
composed of sponge
and filter paper,
soaked in a tank filled
with buffer

23. iBlot Dry Blotting
System
Semi-dry transfer Wet or semi-wet transfer
Transfer buffer
required?
No 100–250 mL, or just enough to
construct a bubble-free sandwich
1–1.5 L, or enough to fill the
transfer tank
Transfer time 7 min, plus 2 min
transfer preparation
45–90 min, plus 70 min
preparation and assembly
1 hr–overnight, plus 50 min
preparation and assembly
Transfer quality •Reproducible and
good transfer
quality for proteins
between 11 and 220
kDa.
Variable and inefficient transfer
quality:
Reduced buffer capacity limits
transfer time, especially for mid-
to large molecular weight proteins
Membrane and filter paper MUST
be cut to exact gel size, otherwise
current will short-circuit around
the edge of the gel
•Reliable and good transfer
quality:
Increase temperature during
blotting, unless buffer is
mixed and cooled during
blotting
•High-current power source
is typically required for 1–2
hour transfer
Supple. equipment
required
None External power supply External power supply
Post-transfer
requirement
None •Wet-soaking filter paper for
clean-up
•Salt deposits on electrodes
require regular maintenance
•Large amount of hazardous
buffer to discard
•Wet-soaking sponges for
clean-up
•Salt deposits on electrodes
require regular maintenance

24. 4. STABILIZATION
• Once the RNA has been transferred to the membrane, it is
immobilized through covalent linkage to the membrane by two
methods.
1. UV crosslinking is one of the most popular methods, using either a
hand-held UV lamp at short wavelength, or a commercial crosslinking
device.
• Shortwave UV light causes the uracil base to become highly reactive
and to form covalent linkages to amine groups on the surface of the
membrane.
• The "auto-crosslink" feature on commercially available, calibrated UV
crosslinkers.
• If a calibrated instrument is not available, it is possible to use standard
laboratory equipment such as transilluminators and handheld
ultraviolet lamps to fix RNA targets to a membrane.
• Care must be taken not to under or overexpose the RNA to UV light —
both of which will decrease hybridization signals. Usually a one minute
exposure with 254 nm light.
• The other common method baking the membrane in an oven at 80°C

25. 2. Baking works by heating the membrane to drive out all
water solubilizing the RNA.
• A large component of RNA is its hydrophobic nucleotide
bases, which make hydrophobic contacts with aromatic
groups on the membrane.
• This interaction is affected by heating in an oven at 80°C
for 15 min.
• The only danger in baking is that the membrane can be
damaged if the heat is not regulated to prevent
temperatures from rising much higher than 100°C.

26. Probes
• Probes for northern blotting are composed of nucleic acids with a
complementary sequence to all or part of the RNA of interest, they can be
DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to
the target sequence.
• RNA probes should be withstand during more rigorous washing steps
preventing some of the background noise.
• Commonly cDNA is created with labelleled primers for the RNA sequence of
interest to act as the probe in the northern blot.
• The probes must be labelled either with radioactive isotopes (32P, 33P, or 35S.
Radioactive labeling provides the most sensitive method for detection,
allowing detection of 0.01 pg.) or with chemiluminescence in which alkaline
phosphatase or horseradish peroxidase (HRP) break down chemiluminescent
substrates producing a detectable emission of light.
• The chemiluminescent labelling can occur in two ways: either the probe is
attached to the enzyme, or the probe is labelled with a ligand (e.g. biotin) for
which the ligand (e.g., avidin or streptavidin) is attached to the enzyme (e.g.
HRP).
• X-ray film can detect both the radioactive and chemiluminescent signals and
chemiluminescent signals are faster, more sensitive, and reduce the health
hazards.

27. Detection radioactive labelled probe (Disadvantages of 32P):
• Short half life (about 2 weeks) means probes must be used immediately, and
the labeling reagent cannot be stored for long.
• Contamination problems: all materials and equipment must be dedicated to
radioactive work only. Regular lab-wide testing for contamination is required.
• Expense of disposal of radioactive waste.
• Must have access to a dark room to set up and develop films.
Nonradioactive detection system are colorimetric, fluorescent and
chemiluminescent
Colorimetric detection generally involves the production of a colored precipitate
which can be seen with the naked eye. In a typical system, the DNA probe
itself is labeled with an antigen such as Digoxigenin and after hybridization to
its target it would be exposed to an anti-digoxigenin antibody conjugated to an
enzyme capable of catalyzing a colorimetric reaction.
Fluorescent detection involves probes which are directly labeled with
fluorophores, or indirectly. For example, if probe is labeled with biotin, it
would be exposed to avidin or streptavidin fluorescent tag and Fluorophores
emit light at an appropriate wavelength.
Chemiluminescence is a combination of these two: an enzymatic reaction that
triggers the release of ordinary visible light.

28. 5. HYBRIDIZATON AND WASHING OF EXCESS PROBS
• Hybridization with radiolabelled or fluorescently labelled
probe.
Prehybridize before hybridization:
• Blocks non-specific sites to prevent the single-stranded probe
from binding just anywhere on the membrane.
Hybridization:
• Incubate membrane with labeled RNA probe with target
sequence: Probe could be lablleled with 32P,
biotin/streptavidin probe.
• Probes for northern blotting are composed of nucleic acids
with a complementary sequence to all or part of the RNA of
interest, they can be DNA, RNA, or oligonucleotides with a
minimum of 25 complementary bases to the target sequence.
• The probes must be labelled either with radioactive isotopes
(32P) or with chemiluminescence.
• Hybridization between probes and the target RNA
• Washing of excess probes

29. 6. VISUALIZATION
Autoradiography:
• Place membrane over X-ray film.
• X-ray film darkens where the fragments are complementary
to the radioactive probes.

31. NORTHEN BLOT APPLICATION
• Northern blots are particularly useful for determining the
specific genes are being expressed at mRNA level.
• Northern blotting allows one to observe a particular gene's
expression pattern between tissues, organs, developmental
stages, pathogen infection, and over the course of treatment
• The technique has been used to show overexpression of
oncogenes and downregulation of tumor-suppressor genes in
cancerous cells when compared to 'normal' tissue, as well as
the gene expression in the rejection of transplanted organ.

32. Comparison of Northern, Southern and Western blotting

33. Thank You