https://doi-org.biblioteca.ibt.unam.mx:8080/10.1038/npg.els.

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Abstract
Northern blotting is a means of identifying a certain RNA species in a complex mixture of RNA.
Commonly used to evaluate qualitatively and quantitatively the expression of a gene, Northern
blotting involves isolation of RNA, size fractionation of the denatured RNA through gel
electrophoresis, transfer of the separated RNA to a membrane, hybridization with a specific
probe, and detection.

The Principle of the Northern Blot

Northern blotting measures the relative amount and size of a specific RNA sequence within
a complex RNA preparation using methods similar to those developed for Southern blotting
of DNA (Southern, 1975). The differences between Northern and Southern blotting are due
to the distinct chemical properties of RNA versus DNA. In Northern blotting, total or
poly(A)+ RNA is isolated from cells or tissues, heat‐denatured to melt secondary structure,
then size fractionated by denaturing gel electrophoresis. The separated RNA is transferred
to a membrane, which is then subjected to hybridization in solution with a labelled RNA or
denatured DNA probe after prehybridization to reduce background (Alwine et al ., 1977).
Since RNA is single‐stranded and available to bind single‐stranded DNA or RNA probes,
the blot is not denatured prior to the hybridization step, as it would be in Southern blotting.
After washing to remove unbound probe, detection is accomplished using label‐specific
methods, i.e. autoradiography for radiolabelled probes or secondary detection systems akin
to Western blotting for nonradioactively labelled probes. The RNA species of interest
presents as a single or several discrete bands, depending upon the nature of the probe and
target RNA and upon the stringency of hybridization (Figure 1). The entire process of
Northern blotting is lengthy, often requiring several days to complete. See also Southern
Blotting and Related DNA Detection Techniques, and Western Blotting:
Immunoblotting
Figure 1
Open in figure viewerPowerPoint
Flowchart indicating the steps of Northern blotting and detection of RNA.

Several molecular biology resource manuals provide excellent comprehensive descriptions

of Northern blotting along with step‐by‐step protocols and recipes (Brown and
Mackey, 1997; Sambrook et al. , 1989). This article describes the general principles of the
technique, including the preparation, separation and blotting of RNA, followed by a
description of hybridization and detection methodologies. It concludes with a brief
discussion of the applications and limitations of Northern blotting and describes several
alternative methods of detecting RNA.

Preparation of RNA
Northern blotting requires the isolation of clean, intact full‐length RNAs. RNA preparations
contaminated with DNA and protein may result in aberrant sample migration or increased
background, whereas partially degraded RNAs will ultimately appear as a smear or as
multiple bands, confounding identification and quantitation. Unfortunately, RNAs are
notoriously difficult to isolate since they are extremely susceptible to digestion by
ribonucleases, a class of ubiquitous, stable enzymes requiring no cofactors. Therefore, most
RNA‐isolation procedures employ as a first step homogenization of the cell or tissue
sample in a denaturing, reducing chemical environment to inactivate ribonucleases. The
careful use of sterile, ribonuclease‐free solutions, glass and plastic ware and the wearing of
gloves during all RNA preparation steps reduce the likelihood of sample degradation. The
treatment of solutions with diethyl pyrocarbonate also helps to eliminate residual
ribonuclease activity. Homogenization of the sample is followed by separation of the RNA
from the rest of the cellular components, then precipitation and purification of the RNA
fraction away from contaminating DNA and small RNA species. See also RNA: Methods
for Preparation, and Ribonucleases

Several methods are commonly used for isolation of RNA from a variety of plant and
animal sources. The source of the RNA, along with sample size and investigator preference
usually dictates which of the most common methods is used. Comprehensive protocols
describing these RNA‐isolation procedures are readily available in standard molecular
biology techniques manuals.

Many RNA‐extraction methods employ homogenization of cells or tissues in phenol to

denature proteins and centrifugation to separate them from nucleic acids which are
subsequently precipitated in the presence of ethanol and sodium or potassium acetate (at a
low pH ∼5.0 to minimize RNA hydrolysis) (Kirby, 1968). A second precipitation removes
DNA, small RNAs, and polysaccharide and proteoglycan material. See also Protein
Unfolding and Denaturants, and Centrifugation Techniques

Guanidinium salts in high concentrations and other denaturing agents dissociate

ribonucleoprotein complexes and are useful in isolating RNA from tissues with high
ribonuclease activity (Chirgwin et al. , 1979). Guanidinium thiocyanate and 2‐
mercaptoethanol are used in combination to reduce and denature ribonucleases and other
proteins. RNA is isolated by high‐speed centrifugation followed by precipitation with
ethanol. A modification of this method involves centrifugation through a caesium chloride
cushion to prevent precipitation of DNA along with the RNA pellet (Glisin et al. , 1974). A
single‐step, guanidine‐based method permits rapid isolation of RNA of sufficient quality
for Northern blotting by employing liquid‐phase separation (Chomczynski and
Sacchi, 1987). Commercial preparations based upon the single‐step method are widely
available and ease preparation time and handling. Several of these methods allow
simultaneous purification of DNA and protein along with RNA.

Homogenization of frozen tissue in 3 mol L−1 LiCl, 6 mol L−1 urea is another method of
obtaining intact RNA (Auffray and Rougeon, 1980). Ribonucleases are inhibited in these
conditions, which also allow for the preparation of RNA largely free of contaminating
DNA, proteins and polysaccharides.

The quantity of RNA recovered using any of these methods may be determined by
measuring the absorbance of the diluted sample at 260 nm and 280 nm. A pure preparation
of RNA will have an A260:A280 ratio of 2.0, while a 40 mg/mL solution of RNA will have
an A260 of 1.

Poly(A )+ RNA selection

Most RNA‐isolation procedures result in the purification of large amounts of ribosomal
RNAs and small RNAs along with messenger RNAs. In order to increase the sensitivity of
Northern blotting, it may be desirable to purify polyadenylated (poly(A)+) messenger RNA
away from other RNA species. Purification of poly(A)+ RNA from total RNA results in a
100‐ to 1000‐fold enrichment, depending on the organism and tissue source. Messenger
RNA‐purification methods are based upon binding of the long 3′ poly(A) tract to oligo‐dT
immobilized on a solid support such as cellulose. Ribosomal RNAs, transfer RNAs and
other RNA species lacking the poly(A) tail will not bind. Typically one packs a small,
RNase‐free column with commercially available oligo‐dT cellulose. The total RNA sample
is heated to disrupt secondary structure and applied to the column in high salt conditions to
promote binding. The unbound RNA that elutes is heated and reapplied to the column
several times to improve yield. The column is then washed extensively with loading buffer
to remove unbound RNAs. Finally, the poly(A)+ RNA is eluted with low‐salt buffer. Often
the procedure must be repeated to eliminate most of the contaminating RNAs. See
also Cellular RNAs: Varied Roles, mRNA Formation: 3′ End, and Affinity
Chromatography

Messenger RNAs with shorter poly(A) tracts cannot be retained on oligo‐dT cellulose, but
can be purified over poly(U) Sepharose. In this case, the poly(A)+ RNA is eluted by
stronger denaturing conditions such as 80–90% formamide or increased temperatures.

Separation of RNA
Northern blotting requires separation of the RNA species by size, usually through agarose
gel electrophoresis. Since RNA can form secondary structures through complementary base
pairing, it must be denatured by heating prior to electrophoresis. Typically, RNA samples
are mixed with loading buffer containing formaldehyde and formamide and heated to 60°C
for several minutes. To keep the RNA denatured, formaldehyde (Lehrach et al. , 1977) or
glyoxal (McMaster and Carmichael 1977) are added to the gel matrix. Use of formaldehyde
does not require recirculation of buffer throughout the electrophoresis run, as does glyoxal
traditionally (McMaster and Carmichael 1977; O'Connor et al ., 1991), but can be
problematic because formaldehyde is volatile, toxic and classified as a carcinogen. A new
method which describes electrophoresis of RNA with glyoxal in the absence of buffer
recirculation may provide a less noxious alternative (Burnett, 1997). Horizontal agarose gel
electrophoresis is used most commonly for Northern blotting, however polyacrylamide gel
electrophoresis can result in greater sensitivity and resolution. See
also RNA Structure, Nucleic Acids: Thermal Stability and Denaturation, and Gel
Electrophoresis: One‐dimensional

Blotting of RNA
Transfer of RNA from the electrophoresis gel to a solid support or membrane is usually
effected by ascending (Southern, 1975) or descending capillary transfer
(Chomczynski, 1992), electroblotting (Smith et al. , 1984), or vacuum blotting (Peferoen et
al. , 1982). In the first instance, the gel is placed between a paper wick and the membrane.
Capillary action draws the high‐salt transfer buffer through the wick, through the gel, and
into the membrane and stacks of paper towels situated above (Figure 2). Efficient transfer
of RNA from gel to membrane is accomplished overnight. Faster although more costly
transfer can be effected using commercially available blotting apparatus. Electroblotting
apparatus permit rapid transfer of RNA to membranes by electrophoresis in a semi‐dry
format or by immersion in a tank filled with transfer buffer. Vacuum blotting apparatus are
typically used with dot blot and slot blot manifolds.
Figure 2
Open in figure viewerPowerPoint
Diagram of Northern blotting by the ascending capillary transfer method of Southern
(Southern, 1975). The agarose gel containing the size‐fractionated RNA (left) is placed on a
stack of blotting paper which acts as a wick to draw the buffer through the gel, through the
membrane and into the blotting paper and paper towels. RNA from the gel becomes
immobilized on the membrane.

Efficiency of transfer can be evaluated by staining the gel with ethidium bromide (EtBr)
and viewing under ultraviolet (UV) light. Conversely, EtBr can be added to the RNA
sample prior to electrophoresis, permitting direct visualization of RNA on the membrane
after transfer by viewing under UV light. If total RNA is electrophoresed in the presence of
EtBr, a photograph of the membrane can serve to evaluate the integrity of individual RNA
samples bands as well as the consistency of loading across the gel by visualization of the
major ribosomal RNAs. Transfer efficiency and loading can also be evaluated by staining
of the membrane with methylene blue. Because methylene blue treatment and destaining
procedures destroy the blot for purposes of hybridization, this should only be done after the
blot has been probed. RNA sizing is often performed by running RNA size standards in the
first lane of the gel, then slicing off the relevant lane of the transferred blot and staining.
See also DNA Intercalation, and Gel‐Staining Techniques – Dyeing to Know It All

Choice of Membranes and Crosslinking

There are three types of membranes available for Northern blotting: nitrocellulose, nylon or
chemically charged. Nitrocellulose binds RNA with high efficiency, although fragments
smaller than 500 nucleotides (nt) bind poorly. Nitrocellulose is also fragile and tends to
become brittle with repeated use. Therefore, nitrocellulose blots can be probed only a few
times. Nylon membranes are thought to confer better sensitivity than nitrocellulose and
bind small RNA species efficiently. Additionally, they are flexible and can be reprobed
several times. Both nitrocellulose and nylon membranes require crosslinking of the
transferred RNA to the membrane, usually by baking, chemical treatment
or UV crosslinking. Chemically charged membranes have lower binding capacity than
nitrocellulose or nylon, however transferred RNAs will bind these membranes covalently
without chemical treatment or UVirradiation. All three types of membranes are compatible
with capillary, electroblotting or vacuum blotting methods of transfer.

Blocking, Hybridization and Washing

After transfer and crosslinking, the blot is prehybridized to coat it and prevent labelled
probe from binding nonspecifically. Failure to block sufficiently can lead to high
background signals. Prehybridization is performed by incubating the blot for 20 min to
several hours at the hybridization temperature using the hybridization solution without
probe. Both prehybridization and hybridization steps require heating and agitation of the
blot and solution; therefore these steps are normally performed in plastic containers or heat‐
sealed bags immersed in shaking, heated waterbaths or in bottles placed in rotating racks
within hybridization ovens.

As reviewed in Bej (see Bej, 1996), kinetics of hybridization are determined by the melting
temperature of the duplex, the composition of the hybridization and wash solutions, and the
temperature. These factors affect both the rate of hybrid formation and hybrid stability.
Buffer conditions, temperature and duration of hybridization therefore should be optimized
for each hybridization reaction. Broadly speaking, one may carry out reactions in
conditions of high stringency for the hybridization of only well‐matched or highly
complementary targets and probes, or low stringency to allow duplexing of probe to targets
with a high degree of mismatch.

Hybridization is generally carried out in aqueous solutions or alternatively in the presence

of formamide to reduce hybridization temperatures. Typical hybridization solutions may
contain salt, sodium dodecylsulfate (SDS), sheared genomic DNA (salmon sperm or
placental) to prevent high background, and high‐molecular‐mass polyethylene glycol or
dextran sulfate and Ficoll to concentrate probe. If probe and target are well matched,
hybridization is performed at high temperatures (65–68°C for aqueous solutions, 42°C in
the presence of 50% formamide). For lower stringency, hybridization is performed in 25%
formamide and/or at lower temperatures (35–42°C). Northern blots are normally hybridized
overnight. See also Nucleic Acid: Hybridization
Posthybridization washes are required to remove unhybridized probe from the solid support
and to dissociate loosely bound probe. Generally, for high stringency, washing is performed
in the presence of low salt and high temperature (e.g. 0.1X SSC, 0.2% SDS at 50°C).
Lower stringency washes are performed in high salt and/or at lower temperatures. See
also Nucleic Acid Structure and Stability: Effects of Salt/Divalent Cations

Types of Hybridization Probes

Broadly, there are two types of probes used in Northern blotting – RNA and DNA probes.
Most commonly, double‐stranded DNA probes are used. Any probe need only be long
enough to be efficiently labelled and to detect and specifically identify the RNA of interest.
Often cDNAs or partial cDNAs are used. DNA probes can be synthesized by PCR
amplification in the presence of radionucleotides or hapten molecules that are incorporated
during DNA synthesis. DNA probes can also be generated by end‐labelling using
polynucleotide kinase or terminal deoxyribonucleotidyltransferase, nick‐translation using
DNase I and DNA polymerase I, or random priming using random hexamers and Klenow
fragment. Double‐stranded DNA probes must be denatured prior to hybridization, typically
by boiling followed by rapid cooling, or by alkali treatment. Once in the hybridization
solution, DNA probes must undergo two competing reactions: hybridization to target RNA
on Northern blots, and hybridization to its complementary strand in solution. In practice,
however, this competition presents no problems. See also Nucleic Acids: General
Properties, and Polymerase Chain Reaction (PCR)

Single‐stranded DNAs such as those generated by M13 phage or even oligonucleotides can
be used as probes as well. These probes have the advantage that there is no competition in
the liquid phase for the complementary probe strand. End‐labelled oligonucleotide probes
(16–30 nt) are useful in the detection of abundant RNAs such as the ribosomal RNAs in
total RNA preparations. See also Nucleotides: Uncommon, Modified and Synthetic

RNA probes, also known as riboprobes, are useful in Northern blotting because
RNA : RNA hybrids associate more tightly than DNA : RNA hybrids, theoretically
increasing sensitivity of detection. RNA probes can also distinguish two transcripts
produced by directional transcription from either strand of a DNA template, since the probe
itself is single‐stranded and will complex only with its complementary sequence. RNA
probes are more cumbersome to prepare, requiring in vitro transcription with labelled
ribonucleotides of DNA templates containing RNA polymerase‐recognition sites. RNA
probes are themselves vulnerable to ribonuclease. After denaturation to reduce secondary
structure, however, RNA probes have no competition with self in the liquid phase of
hybridization, freeing all probe molecules to hybridize with the target. See
also Transcription and Translation In Vitro

Whether RNA or DNA probes are used, the investigator has a choice of labelling agents.
Traditionally, radiolabelled nucleotides (generally 32P) are used for Northern blotting
applications. Radiolabelled probes are sensitive enough to detect less than 1 pg to 1–10 fg
of target RNA and are visualized by exposure of the blot to autoradiography film or
phosphor imager cassette. Recently, nonradioactive alternatives have been made available,
including digoxigenin or biotin labelling moieties (for a comprehensive review, see
Düring, 1993). Visualization of such probes requires secondary detection systems that often
involve antibody hybridization, washing and detection steps. Biotin‐labelled probes, for
example, are normally detected by incubation with an enzyme‐conjugated antibiotin
antibody followed by a colorimetric, chemiluminescent or chemifluorescent reaction
generated in a process similar to Western blotting. Colorimetric reactions result in visible
bands on the membrane itself. Chemiluminescence is detected indirectly by exposure to
autoradiography film or imaging system, while chemifluorescence requires fluorometry in
charge‐coupled device (CCD) cameras or other imaging instruments. Such nonradioactive
systems have the advantage of eliminating exposure to and disposal of the high‐energy
beta‐emitter 32P, but require multiple steps after hybridization and washing. In recent
developments, these latter steps have been obviated by direct labelling of DNA probes with
alkaline phosphatase for chemiluminescent or chemifluorescent detection or with
fluorochromes for direct detection. See also Radioisotopes and Liquid Scintillation
Counting, Autoradiography and
Fluorography, Phosphorimager, Biotin, Immunoassay, Immunofluorescence,
and Image Analysis Workstations

Application of RNA Blotting

Northern blotting reports the steady state level of gene expression in cells, tissues or
organisms. As such, it is useful in the initial characterization of a gene, for example in
identifying tissues, developmental stages or cells in which the gene is expressed. One can
examine differences in gene expression between samples or over time by isolating samples
at increasing timepoints or after experimental manipulation. One must caution, however,
that comparisons can only be made among samples on a single blot and not across two or
more different blots, as electrophoresis, blotting, hybridization, washing and exposure
conditions all result in variations in signal intensity. See also Gene Identification and
Isolation, and Gene Expression - External and Internal

One means of quantifying differences in signal levels between samples is to normalize the
signal for the gene of interest (calculated from phosphor imager data or from densitometry
of autoradiographic film bands) against the signal seen for a gene that is uniformly
expressed in all samples. Investigators often choose so‐called ‘housekeeping genes’ such as
actin, aldolase or glyceraldehyde phosphate dehydrogenase (GAPDH) for this purpose. In
most cases, blots are probed first for the gene of interest. Then, once a satisfactory signal is
obtained, the blot is stripped (usually by boiling in low‐salt, detergent‐containing solutions)
and reprobed for the housekeeping gene. In this manner, a single blot can be probed several
times for different transcripts. See also Housekeeping Genes

Limitations of RNA Blotting Methods

Certain limitations are inherent in Northern blotting. Some result from the amount and
quality of RNA used. Since Northern blotting requires full‐length, intact RNA for
interpretation, this technique is not useful for examining partially degraded RNA samples.
Because isolation of RNA depends upon clean, rapid processing of fresh or flash‐frozen
samples, unpreserved samples and many clinical and field samples are not amenable to
Northern analysis. New commercially available products, such as RNALater (Ambion,
Austin, Texas), that permit storage of samples in an RNA‐preserving buffer at room, cold
or freezing temperatures may make sample preservation less of a chore. Northern analysis
also requires a minimum sample size or amount of starting RNA for sensitive detection of a
message, usually 1–5 μg of poly(A)+ or 10–30 μg total RNA. Very small samples,
therefore, may be more easily studied using another method.

A signal obtained by Northern blotting represents the steady state level of that transcript in
a given sample. An increase in signal from one sample to the next in a given experiment
reflects only in increase in total transcript levels, failing to discriminate stabilization of
transcripts (inhibition of degradation) from de novo synthesis of transcripts. Interpretation
of Northern results is also complicated by the fact that closely related transcripts can
hybridize to a given probe, although this limitation can be minimized by judicious choice of
probe and increasing stringency of hybridization and washing conditions. Northern analysis
is also limited by the nature of the sample; localization of cells expressing the gene of
interest within a tissue or other heterogeneous samples is impossible using this method.
Finally, due to the size of the gels and membranes and multiple manipulations required,
Northern blotting is not particularly amenable to automation or high‐throughput screening.

Other RNA Detection Methods (not including

RT -PCR )
Other RNA detection methods are useful in situations in which Northern blotting is
impossible or otherwise undesirable. These methods include dot/slot blotting, ribonuclease
protection assay and in situ hybridization, which are discussed below, along with reverse
transcription polymerase chain reaction, (RT‐PCR) and high density oligonucleotide array
analysis, which are discussed elsewhere. See also Polymerase Chain Reaction (PCR):
Specialised Reactions

When large numbers of samples are to be screened for the presence of a particular RNA
species, ‘dot blotting’ or ‘slot blotting’ may be advantageous. In these procedures, RNA is
transferred to a solid support without prior size fractionation. Instead, RNA samples are
applied to a geometric grid of round (dot) or rectangular (slot) wells and vacuum‐blotted
directly to a membrane. Common dot and slot blotting apparatus provide for 96 or more
samples and are often formatted for use with a multichannel pipettor, thereby facilitating
throughput. Another advantage of these techniques is the detection of even partially
degraded RNAs which would appear as a smear or as lower molecular weight bands in
Northern blotting. Disadvantages include high background due to nonspecific hybridization
to other RNAs and lack of specificity due to cross‐hybridization to related RNAs. Thus
confirmation of positive results by subsequent Northern blotting of selected samples should
be considered.

When sample size is limiting or when target RNAs may be partially degraded, ribonuclease
protection assays (RPAs) provide an alternative to Northern blotting. In this technique, a
solution of sample RNA containing the species of interest is hybridized to an excess of
labelled complementary RNA probe. As in Northern blotting, the probe may be either
radioactive or nonradioactive, with the latter requiring a secondary detection system.
Posthybridization, the entire sample is precipitated and subjected to digestion with RNase
H, which selectively degrades single‐stranded RNA. In this manner, all nonhybridized
probe and target RNA will be degraded. The sample is then subjected to denaturing
polyacrylamide gel electrophoresis, followed by drying and signal detection. The amount of
protected probe is used as a measure of the amount of target RNA present in the starting
sample. The signal for the RNA of interest is typically normalized to that of a control
transcript. RPAs are generally more sensitive than Northern blotting methods because
solution hybridization has the fastest kinetics and because all of the target RNA is available
for hybridization unlike some of the RNA bound to a membrane. Another advantage
of RPAs is the ability to detect multiple RNA species in a single sample through the use of
a mixture of probes, each of a discrete, known length so as to be distinguishable from all
others. Such use of several probes is referred to as multiplex RPA. Several commercial
suppliers sell multiplex RPA kits for measuring expression of families or classes of genes.
These kits can provide a rapid evaluation of gene expression, however many investigators
choose to confirm those findings by Northern analysis. Major drawbacks of RPAs are the
added effort of probe template preparation, the extensive handling and manipulation of
radioactive samples, the challenge of preventing RNA degradation during sample and probe
preparation, the use of more cumbersome and toxic polyacrylamide gels rather than agarose
gels, and the need for additional equipment such as a gel dryer.

Another obvious limitation of Northern blotting is the inability to localize a particular RNA
species within a given sample. Identification of the cellular source of the RNA signal,
therefore, is limited by one's ability to separate expressing cells and tissues from
nonexpressing cells and tissues. For fine localization of RNA to tissues within organisms,
cells within tissues, or even within cellular compartments, one may instead use the
technique of in situ hybridization. This technique permits solution hybridization of a
labelled RNA probe to its cognate target within a fixed tissue or organism, or on a fixed
tissue section or cell preparation. The tissue is prepared for in situ hybridization by rapid
harvesting and fixation, chemical or enzymatic permeabilization, and prehybridization.
Both prehybridization and hybridization are performed in similar solutions to those used for
Northern blotting. Labelled complementary RNA probes are synthesized by in
vitro transcription from DNA templates and are subsequently denatured and added to the
hybridization solution. Alternatively, labelled oligonucleotides can be used. Extensive
washes and, in the first case, treatments with RNase to eliminate unhybridized probe are
employed to reduce background signals. When radionuclides such as 35S, 32P or 33P are used
to make probes, the hybridization signal is detected either by direct exposure of the entire
tissue section to autoradiographic film or by coating of the tissue section with photographic
emulsion. Probes labelled with fluorochrome‐tagged ribonucleotides can be visualized
directly under a fluorescence microscope. Alternatively, fluorochromes and other moieties
such as biotin and digoxgenin can be detected with an enzyme‐conjugated antibody against
the label. Often this is accomplished by incubation with an alkaline phosphatase labelled
antibody followed by extensive washes and colour development in an NBT‐BCIP (4‐nitro
blue tetrazolium chloride, 5‐bromo‐4‐chloro‐3‐indolyl‐phosphate 4‐toluidine salt) solution.
See also In Situ Hybridization, and Fluorescence in situ Hybridization

Summary
Although a variety of methods are currently available for detecting specific RNA species
within a complex mixture, the commonly used procedure known as Northern blotting
provides a simple means of evaluating the abundance and size of a given RNA. This basic
molecular biology technique involves separation of RNA by size on an agarose gel, transfer
of the separated RNA species to a membrane, and detection of a given RNA sequence using
a specific hybridization probe.