Northern Blotting
Content
Introduction .............................................................................................................................................2
Materials for northern blot .......................................................................................................................3
Method for northern blot .........................................................................................................................4
Prepare RNA Sample for Loading ..........................................................................................................4
Visualise integrity of RNA by UV illiumination/EtBr staining .............................................................5
Transfer to nylon membrane ...................................................................................................................5
Hybridization ...........................................................................................................................................7
1
Northern Blotting
INTRODUCTION
Northern blot technique is very useful in the analysis of RNA molecules. This analysis is the RNA
blotting like southern blot. Generally this technique involves the isolation of the RNA molecules from
sources of interest and the separation of the isolated RNA by agarose or polyacrylamide gel
electrophoresis, the separate RNA in the gel is then transferred to a solid matrix such as filter paper
through the blotting process. Northern blot analysis allows the detection and quantification of
specific RNA species from a particular cell type. Isolated RNA is electrophoresed through an
agarose/formaldehyde gel which separates the RNA species by size. The faster migrating RNA
fragments are the smallest, however, the distance of migration is not linear, rather it is inversely
proportional to the size RNA molecule. When RNA has separated following electrophoresis, it is
stained with ethidium bromide and visualised using ultra violet light. To probe for a specific
mRNA species by northern blot, it is first necessary to transfer the RNA from the
agarose/formaldehyde gel to a nylon membrane. RNA is detected by hybridisation using a
labelled probe. The probe is a DNA or RNA molecule which is chemically or radioactively
labelled.
2
Northern Blotting
MATERIALS FOR NORTHERN BLOT
RNase free water
Agarose
10X running buffer (0.2 M MOPS pH 7.0, 10 mM EDTA)
Formaldehyde
Loading dye
Formamide
Ethidium bromide
0.5 M iodoacetamide
Microfuge
UV transilluminator
Hybond N (Nylon) membrane
20 x SSC (0.3M Na3citrate, 3M NaCl)
Saran wrap
Glass plate
Whatmann 3MM paper
Glass as weight for blot
Sponge soaked in 20 x SSC
3
Northern Blotting
METHOD FOR NORTHERN BLOT
1. Prepare RNA Sample for Loading
a. Add 10-20 µg of RNA to a sterile eppendorf tube
b. Adding formamide


Formamide lowers annealing temperature of the probe-RNA interaction,
Preventing RNA degradation by high temperatures
c. Adding formaldehyde (2.2 M)

Formaldehyde is used for denaturing agent for the RNA to limit secondary structure.
d. Adding MOPS

MOPS is the most commonly used buffer for RNA gels due to its high buffering capacity
at pH 7.0
e. Adding loading dye
f. Pipetting the solution
2. Load samples into the wells and run at 100 volts for 2 hours

The RNA samples are most commonly separated on agarose gels
containing formaldehyde as a denaturing agent for the RNA to limit secondary structure
4
Northern Blotting
3. Visualise integrity of RNA by UV illiumination/EtBr staining

The gels can be stained with ethidium bromide (EtBr) and viewed under UV light to
observe the quality and quantity of RNA before blotting.
4. Transfer to nylon membrane

A nylon membrane with a positive charge is the most effective for use in northern blotting
since the negatively charged nucleic acids have a high affinity for them
a. Set up transfer
b. Place whatman 3 mm filter paper
c. Add transfer buffer
d. Soak the paper
e. Place filter paper
f. Remove any air bubbles
5
Northern Blotting
g. Place the gel
h. Put a nylon membrane on the gel
i. Flood the membrane with transfer buffer
j. Remove any air bubbles
k. Cover with whatman 3 mm filter paper
l. Cover with plastic wrap
m. Stack paper towels above the gel
n. Allow transfer to go overnight
6
Northern Blotting
5. Disassemble the transfer system
a. Remove paper towel and filter papers
b. Remove paper towel and filter papers
c. Fix the RNA to nylon membranes using UV crosslinker
6. Hybridization
a. Prehybridize membrane in prehybridize buffer
b. Incubate for 2-4 hours
c. Discard the prehybridizied buffer
d. Add hybridisation buffer
7
Northern Blotting
7. Addition of 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.
a. Incubate overnight in hybridisation chamber
b. After incubation, discard the solution
c. Add wash solution
d. Incubate at 52 C for 30 minutes in hybridisation chamber (repeat 3 times)
e. Remove the membrane
8
Northern Blotting
8. Membrane is ready to expose to film
9