Northern blot

are fragile and the probes are unable to enter the matrix,
the RNA samples, now separated by size, are transferred
to a nylon membrane through a capillary or vacuum blotting system.
weight
Glass plate

paper towels
capillary blotting paper
Agarose gel
Transfer buer

Capillary blotting system setup for the transfer of RNA from an

electrophoresis gel to a blotting membrane.
Flow diagram outlining the general procedure for RNA detection
by northern blotting.

A nylon membrane with a positive charge is the most

eective for use in northern blotting since the negatively charged nucleic acids have a high anity for them.
The transfer buer used for the blotting usually contains formamide because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating
the need for high temperatures, which could cause RNA
degradation.[9] Once the RNA has been transferred to
the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. After a probe
has been labeled, it is hybridized to the RNA on the
membrane. Experimental conditions that can aect the
eciency and specicity of hybridization include ionic
strength, viscosity, duplex length, mismatched base pairs,
and base composition.[10] The membrane is washed to
ensure that the probe has bound specically and to prevent background signals from arising. The hybrid signals
are then detected by X-ray lm and can be quantied
by densitometry. To create controls for comparison in a
northern blot, samples not displaying the gene product of
interest can be used after determination by microarrays
or RT-PCR.[10]

The northern blot is a technique used in molecular biology research to study gene expression by detection of
RNA (or isolated mRNA) in a sample.[1][2]
With northern blotting it is possible to observe cellular control over structure and function by determining
the particular gene expression rates during dierentiation and morphogenesis, as well as in abnormal or diseased conditions.[3] 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. The term 'northern blot' actually refers specically to the capillary transfer of RNA from the electrophoresis gel to the blotting
membrane. However, the entire process is commonly referred to as northern blotting.[4] The northern blot technique was developed in 1977 by James Alwine, David
Kemp, and George Stark at Stanford University.[5] Northern blotting takes its name from its similarity to the rst
blotting technique, the Southern blot, named for biologist
Edwin Southern.[1] The major dierence is that RNA,
rather than DNA, is analyzed in the northern blot.[6]

1.1 Gels
The RNA samples are most commonly separated on
agarose gels containing formaldehyde as a denaturing
agent for the RNA to limit secondary structure.[10][11]
The gels can be stained with ethidium bromide (EtBr) and
viewed under UV light to observe the quality and quantity of RNA before blotting.[10] Polyacrylamide gel electrophoeresis with urea can also be used in RNA separation but it is most commonly used for fragmented RNA
or microRNAs.[12] An RNA ladder is often run along-

Procedure

A general blotting procedure[4] starts with extraction of

total RNA from a homogenized tissue sample or from
cells. Eukaryotic mRNA can then be isolated through
the use of oligo (dT) cellulose chromatography to isolate
only those RNAs with a poly(A) tail.[7][8] RNA samples
are then separated by gel electrophoresis. Since the gels
1

RNA 2

RNA 1

RNA
Ladder

ADVANTAGES AND DISADVANTAGES

searchers prefer the chemiluminescent signals because

they are faster, more sensitive, and reduce the health hazards that go along with radioactive labels.[15] The same
membrane can be probed up to ve times without a signicant loss of the target RNA.[9]

2 Applications
Formaldehyde gel (1%) with RNA samples run at 100V for 1 hour in 1x MOPS buffer.

RNA run on a formaldehyde agarose gel to highlight the 28S (top

band) and 18S (lower band) ribosomal subunits.

Northern blotting allows one to observe a particular

genes expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment.[8][14][16] The
technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in
cancerous cells when compared to 'normal' tissue,[10] as
well as the gene expression in the rejection of transplanted organs.[17] 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 nding.[17] The
expression patterns obtained under given conditions can
provide insight into the function of that gene. Since the
RNA is rst 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.[7][13] 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.[1]

side the samples on an electrophoresis gel to observe the

size of fragments obtained but in total RNA samples the
ribosomal subunits can act as size markers.[10] Since the
large ribosomal subunit is 28S (approximately 5kb) and
the small ribosomal subunit is 18S (approximately 2kb)
two prominent bands appear on the gel, the larger at close
BlotBase is an online database publishing northern blots.
to twice the intensity of the smaller.[10][13]
BlotBase has over 700 published northern blots of human and mouse samples, in over 650 genes across more
than 25 dierent tissue types.[3] Northern blots can be
1.2 Probes
searched by a blot ID, paper reference, gene identier,
or by tissue.[3] The results of a search provide the blot
Probes for northern blotting are composed of nucleic
ID, species, tissue, gene, expression level, blot image (if
acids with a complementary sequence to all or part
available), and links to the publication that the work origof the RNA of interest, they can be DNA, RNA, or
inated from.[3] This new database provides sharing of inoligonucleotides with a minimum of 25 complemenformation between members of the science community
tary bases to the target sequence.[4] RNA probes (ribothat was not previously seen in northern blotting as it
probes) that are transcribed in vitro are able to withwas in sequence analysis, genome determination, protein
stand more rigorous washing steps preventing some of
structure, etc.
the background noise.[10] Commonly cDNA is created
with labelled primers for the RNA sequence of interest
to act as the probe in the northern blot.[14] The probes
must be labelled either with radioactive isotopes (32 P) or 3 Advantages and disadvantages
with chemiluminescence in which alkaline phosphatase
or horseradish peroxidase (HRP) break down chemilu- Analysis of gene expression can be done by several difminescent substrates producing a detectable emission of ferent methods including RT-PCR, RNase protection aslight.[15] The chemiluminescent labelling can occur in two says, microarrays, RNA-Seq, serial analysis of gene exways: either the probe is attached to the enzyme, or the pression (SAGE), as well as northern blotting.[3][4] Miprobe is labelled with a ligand (e.g. biotin) for which croarrays are quite commonly used and are usually conthe ligand (e.g., avidin or streptavidin) is attached to the sistent with data obtained from northern blots; however,
enzyme (e.g. HRP).[10] X-ray lm can detect both the at times northern blotting is able to detect small changes
radioactive and chemiluminescent signals and many re- in gene expression that microarrays cannot.[18] The ad-

3
vantage 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.[16][18]
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).[4] 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.[10] Compared to RT-PCR, northern blotting
has a low sensitivity, but it also has a high specicity,
which is important to reduce false positive results.[10]
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.[10]
For northern blotting for the detection of
acetylcholinesterase mRNA the nonradioactive technique was compared to a radioactive technique and
found as sensitive as the radioactive one, but requires no protection against radiation and is less time
consuming.[19]

Reverse northern blot

Researchers occasionally use a variant of the procedure

known as the reverse northern blot. In this procedure, the
substrate nucleic acid (that is axed to the membrane) is
a collection of isolated DNA fragments, and the probe is
RNA extracted from a tissue and radioactively labelled.
The use of DNA microarrays that have come into
widespread use in the late 1990s and early 2000s is more
akin to the reverse procedure, in that they involve the use
of isolated DNA fragments axed to a substrate, and hybridization with a probe made from cellular RNA. Thus
the reverse procedure, though originally uncommon, enabled northern analysis to evolve into gene expression
proling, in which many (possibly all) of the genes in an
organism may have their expression monitored.

See also
Southern blot
Western blot
Northwestern blot
Far-Eastern blotting

Far-western blotting
Eastern blot
Dierential display

6 References
[1] Alberts, B., Johnson, A., Lewis, J. Ra, M., Roberts, K.,
Walter, P. 2008. Molecular Biology of the Cell, 5th ed.
Garland Science, Taylor & Francis Group, NY, pp 538
539.
[2] Kevil, C. G., Walsh, L., Laroux, F. S., Kalogeris, T., Grisham, M. B., Alexander, J. S. (1997) An Improved, Rapid
Northern Protocol. Biochem. and Biophys. Research
Comm. 238:277279.
[3] Schlamp, K.; Weinmann, A.; Krupp, M.; Maass,
T.; Galle, P. R.; Teufel, A. (2008). BlotBase: A
northern blot database. Gene. 427 (12): 4750.
doi:10.1016/j.gene.2008.08.026. PMID 18838116.
[4] Trayhurn, P. (1996) Northern Blotting. Pro. Nutrition
Soc. 55:583589.
[5] Alwine JC, Kemp DJ, Stark GR (1977). Method for
detection of specic RNAs in agarose gels by transfer
to diazobenzyloxymethyl-paper and hybridization with
DNA probes. Proc. Natl. Acad. Sci. U.S.A. 74 (12):
53504. doi:10.1073/pnas.74.12.5350. PMC 431715 .
PMID 414220.
[6] Bor, Y.C.; Swartz, J.; Li, Y.; Coyle, J.; Rekosh, D.; Hammarskjold, Marie-Louise (2006). Northern Blot analysis
of mRNA from mammalian polyribosomes. Nature Protocols. doi:10.1038/nprot.2006.216.
[7] Durand, G. M.; Zukin, R. S. (1993). Developmental Regulation of mRNAs Encoding Rat Brain
Kainate/AMPA Receptors: A Northern Analysis Study.
J. Neurochem. 61 (6): 22392246. doi:10.1111/j.14714159.1993.tb07465.x. PMID 8245974.
[8] Mori, H.; Takeda-Yoshikawa, Y.; Hara-Nishimura, I.;
Nishimura, M. (1991). Pumpkin malate synthase
Cloning and sequencing of the cDNA and Northern
blot analysis. Eur. J. Biochem. 197 (2): 331
336. doi:10.1111/j.1432-1033.1991.tb15915.x. PMID
1709098.
[9] Yang, H.; McLeese, J.; Weisbart, M.; Dionne, J.L.; Lemaire, I.; Aubin, R. A. (1993). Simplied
high throughput protocol for Northern hybridization. Nucleic Acids Research. 21 (14): 33373338.
doi:10.1093/nar/21.14.3337. PMC 309787 . PMID
8341618.
[10] Streit, S.; Michalski, C. W.; Erkan, M.; Kleef, J.; Friess,
H. (2009). Northern blot analysis for detection of RNA
in pancreatic cancer cells and tissues. Nature Protocols. 4 (1): 3743. doi:10.1038/nprot.2008.216. PMID
19131955.

[11] Yamanaka, S.; Poksay, K. S.; Arnold, K. S.; Innerarity, T. L. (1997). A novel translational repressor
mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB
mRNA-editing enzyme. Genes Dev. 11 (3): 321333.
doi:10.1101/gad.11.3.321. PMID 9030685.
[12] Valoczi, A., Hornyik, C., Varga, N., Burgyan, J., Kauppinen, S., Havelda, Z. (2004) Sensitive and specic detection of microRNAs by northern blot analysis using LNAmodied oligonucleotide probes. Nuc. Acids Research.
32: e175.
[13] Gortner, G.; Pfenninger, M.; Kahl, G.; Weising, K.
(1996). Northern blot analysis of simple repetitive sequence transcription in plants. Electrophoresis. 17
(7): 11831189. doi:10.1002/elps.1150170702. PMID
8855401.
[14] Liang, P. Pardee, A. B. (1995) Recent advances in dierential display. Current Opinion Immunol. 7: 274280.
[15] Engler-Blum, G.; Meier, M.; Frank, J.; Muller, G.
A. (1993). Reduction of Background Problems in
Nonradioactive Northern and Southern Blot Analysis Enables Higher Sensitivity Than 32P-Based Hybridizations. Anal. Biochem. 210 (2): 235244.
doi:10.1006/abio.1993.1189. PMID 7685563.
[16] Baldwin, D., Crane, V., Rice, D. (1999) A comparison of
gel-based, nylon lter and microarray techniques to detect
dierential RNA expression in plants. Current Opinion in
Plant Biol. 2: 96103.
[17] Utans, U.; Liang, P.; Wyner, L. R.; Karnovsky, M. J.;
Russel, M. E. (1994). Chronic cardiac rejection: Identication of ve upregulated genes in transplanted hearts by
dierential mRNA display. Proc. Natl. Acad. Sci. USA.
91 (14): 64636467. doi:10.1073/pnas.91.14.6463.
PMC 44222 . PMID 8022806.
[18] Taniguchi, M.; Miura, K.; Iwao, H.; Yamanaka, S. (2001).
Quantitative Assessment of DNA Microarrays Comparison with Northern Blot Analysis. Genomics. 71 (1):
3439. doi:10.1006/geno.2000.6427. PMID 11161795.
[19] Kreft, K., Kreft, S., Komel, R., Grubi, Z. (2000). Nonradioactive northern blotting for the determination of
acetylcholinesterase mRNA. Pgers Arch Eur J Physiol, 439:R66-R67

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