PAPER OF BIOMOLECULES

NORTHERN BLOTING

By :
Naseer Ahmed
17728259001
Chemistry Education B

PROGRAM STUDI PENDIDIKAN KIMIA

PROGRAM PASCASARJANA
UNIVERSITAS NEGERI YOGYAKARTA
2017
 PAPER OF BIOMOLECULES
NORTHERN BLOTING
The northern blot, or RNA blot, is a
Northern Blots are named after their big brother: The Southern
technique used in molecular biolo-
blot. Southern blots are a method of detecting DNA and named after the
gy research to study gene expression by
surname of their inventor Ed Southern. A typical Southern blot experi-
detection of RNA (or isolated mRNA) in
ment goes as follows: 1) run a DNA gel, 2) transfer gel contents onto a
a sample.
membrane, 3) hybridize said membrane with radioactive DNA probe, 4)
With northern blotting it is possible to wash off unbound radioactive probe, and 5) detect radioactive probe.
observe cellular control over structure and
Northern Blots are done the same way as Southern blots but RNA
function by determining the particular
gene expression rates during differentia- is detected instead of DNA. So when invented the RNA method was
tion and morphogenesis, as well as in ab- named “Northern blot” as an homage to the original nucleic acid detec-
normal or diseased conditions. Northern tion method. And the naming tradition just continued with Western
blotting involves the use blots.
of electrophoresis to separate RNA sam-
ples by size, and detection with NORTHERN BLOT TECHNIQUE:
a hybridization probe complementary to
part of or the entire target sequence. The
From this diagram we can easily understand the process of northern tech-
term 'northern blot' actually refers specifi-
cally to the capillary transfer of RNA nique .
from the electrophoresis gel to the blot-
ting membrane. However, the entire pro-
cess is commonly referred to as northern
blotting. The northern blot technique was
developed in 1977 by James Alwine, Da-
vid Kemp, and George Stark at Stanford
University. Northern blotting takes its
name from its similarity to the first blot-
ting technique, the Southern blot, named
for biologist Edwin Southern.
The major difference is that RNA,
rather than DNA, is analyzed in the north-
ern blot After all, it is more common these
days to detect and quantify RNA with
quantitative real time (qRT)-PCR than
with Northern Blots. Northern Blots fell
out of favor for two reasons: The per-
ceived difficulty of working with RNA
and because most people don’t like work-
ing with radioactivity. Yet, qRT-PCR is
prone to false positives and negatives, and
reviewers may require Northern Blot con-
firmation of your qRT-PCR results. So
sometimes Northern Blots are a necessary
evil. However, because of its unpopular
nature finding.
 Step 2 :
How to do a Northern Blot Transfer & Link

After successfully separating your

RNA it is now time to transfer
Make RNA your RNA to a nitrocellulose or
nylon membrane, and crosslink it
Northern blots got a bad rap from the days when working with RNA was as hard as there to prevent your RNA from
it got in molecular biology. This really isn’t the case anymore. There are numerous washing away in subsequent
RNA isolation kits on the market now, and lots of RNAse inhibitors available. So steps. To do this you need to ex-
don’t let the idea of working with RNA scare you. With basic precautions, such as pose your membrane to UV light
cleaning the mold from your workbench and wearing gloves, isolating and main- and you will have to calibrate
taining the integrity of your RNA is easy. your UV lamp – cross linker ma-
chines usually have default pro-
grams, just like microwaves. You
Separate & Transfer RNA can also bake your nitrocellulose
membranes in an oven, 30
Step 1: Gel. minutes to 2 hours at 80°C, to fix
your RNA in place
To separate RNA by size a simple TBE – polyacrylamide – urea gel works
well. To learn more about how to do these gels, check out the video on Denaturing
Urea Polyacrylamide Gel Electrophoresis by Summer et al. The RNA samples are most
commonly separated on agarose gels containing formaldehyde as a denaturing agent for Step 3: Stain
the RNA to limit secondary structure. The gels can be stained with ethidium bro-
mide (EtBr) and viewed under UV light to observe the quality and quantity of RNA
before blotting. Polyacrylamide gel electrophoresis with urea can also be used in RNA After you have cross-
separation but it is most commonly used for fragmented RNA or microRNAs. An RNA linked, it is safe to stain the
ladder is often run alongside the samples on an electrophoresis gel to observe the size of membrane with methylene
fragments obtained but in total RNA samples the ribosomal subunits can act as size blue. The position of rRNA
markers blobs, as revealed by the
methylene blue, will then
give you benchmark for esti-
mation where your mRNA
should be. You can also
use RNA markers, but these
are usually not worth the
bother as they degrade quick-
ly and run strangely. Instead
you can estimate where your
mRNA should be relative to
the dyes in the gel and to
the ribosomal RNAs you will
see after staining.
Pro tip:, it’s worth transfer-
ring your whole gel to the
membrane and hybridizing
the full-length membrane in
case your RNA runs differ-
ently than your estimate
Make RNA Probes
Hybridize RNA

Making the probes for your Northern blot is easy as well. And can be The hybridization steps in Nothern
done one of two ways: You can label a DNA oligo that is complementary to Blot follow the same route as for So-
your RNA or 2) You can random labela PCR product homologous to your thern Blot. You pre-hybridize your
sequence. When provided by a manufacturer pre-labelled short oligos can be membrane in the same buffer your
randomly incorporated across the length of your probe during a PCR reaction. probe is in, usually sodium citrate
You can then use a column, which is based on the same principle as the col- buffer (SSC), then add the buffer
umn for PCR cleanup, to separate your labelled, reusable probe from the free with probe and incubate. And I rec-
nucleotides. ommend that you do not faff about
with making Denhardt’s solution
For either of these methods you have a choice of labels. You can stay (which you can buy) for your hybridi-
true to tradition and use a radioactive label, or you can also use non- zation buffer. Making and hybridiz-
radioactive, fluorescently labelled nucleotides. The benefit of using radioac- ing in Church buffer is simple and in
tive labels are that they are less sensitive to the reaction conditions and detec- my experience, works just as well.
tion, but working with radioactivity is not without its risks and paperwork! After hybridization you will then
This makes fluorescent labels more appealing but they do have a reputation of wash the membrane with a high salt
more miss than hit, because they are complicated organic molecules, unlike SSC buffer several times to get rid of
basic radioactive atoms. the unbound probe.
Probes for northern blotting are composed of nucleic acids with a comple-
mentary 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 (riboprobes) that are transcribed in vitro are
able to withstand more rigorous washing steps preventing some of the back-
ground noise. Commonly cDNA is created with labelled 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) or
with chemiluminescence in which alkaline phosphatase or horseradish peroxi-
dase (HRP) break down chemiluminescent substrates producing a detectable
emission of light.[16] The chemiluminescent labelling can occur in two ways:
either the probe is attached to the enzyme, or the probe is labelled with a lig-
and (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 many researchers prefer the chemiluminescent
signals because they are faster, more sensitive, and reduce the health hazards
that go along with radioactive labels. The same membrane can be probed up
to five times without a significant loss of the target RNA

Detect RNA

You have a number of options to detect your bound radioactive

probes, starting with X-ray film (a retro finish to a retro method!) and
ending with digital imaging system.
In summary, these days Northern Blot is not as difficult as it
used to be. But as with any method, don’t get discouraged if it doesn’t
work the first time. Just plow on and think of the molecular biology
bragging rights. No one will question your molecular biology cred if you
can perform a Southern, Nothern and Western blots
 Applications:
Northern blotting allows one to observe a particular gene's expres-
sion pattern between tissues, organs, developmental stages, environmental
stress levels, pathogen infection, and over the course of treatment. The
technique has been used to show overexpression of oncogenes and down-
regulation of tumor-suppressor genes in cancerous cells when compared
to 'normal' tissue as well as the gene expression in the rejection of trans-
planted organs.
If an upregulated gene is observed by an abundance of mRNA on
the northern blot the sample can then be sequenced to determine if the
gene is known to researchers or if it is a novel finding. The expression
patterns obtained under given conditions can provide insight into the
function of that gene. Since the RNA is first separated by size, if only one
probe type is used variance in the level of each band on the membrane
can provide insight into the size of the product, suggesting alternative
splice products of the same gene or repetitive sequence motifs.
The variance in size of a gene product can also indicate deletions
or errors in transcript processing. By altering the probe target used along
the known sequence it is possible to determine which region of the RNA
is missing.
BlotBase is an online database publishing northern blots. BlotBase has
over 700 published northern blots of human and mouse samples, in over
650 genes across more than 25 different tissue types. Northern blots can
be searched by a blot ID, paper reference, gene identifier, or by tis-
sue. The results of a search provide the blot ID, species, tissue, gene, ex-
pression level, blot image (if available), and links to the publication that
the work originated from.
This new database provides sharing of information between mem-
bers of the science community that was not previously seen in northern
blotting as it was in sequence analysis, genome determination, protein
structure, etc.
Written By :
Naseer Ahmed
17728259001
Chemistry Education B
Advantages and disadvantages:
Analysis of gene expression can be done by
several different methods including RT-PCR,
RNase protection assays, microarrays, RNA-
Seq, serial analysis of gene expression
(SAGE), as well as northern blotting. Microar-
rays are quite commonly used and are usually
consistent with data obtained from northern
blots; however, at times northern blotting is
able to detect small changes in gene expression
that microarrays cannot. The advantage that
microarrays have over northern blots is that
thousands of genes can be visualized at a time,
while northern blotting is usually looking at
one or a small number of genes.
A problem in northern blotting is often
sample degradation by RNases (both endoge-
nous to the sample and through environmental
contamination), which can be avoided by prop-
er 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 re-
searcher, since formaldehyde, radioactive ma-
terial, ethidium bromide, DEPC, and UV light
are all harmful under certain exposures. Com-
pared to RT-PCR, northern blotting has a low
sensitivity, but it also has a high specificity,
which is important to reduce false positive re-
sults.
The advantages of using northern blotting
include the detection of RNA size, the observa-
tion of alternate splice products, the use of
probes with partial homology.