![Northern blot diagram](https://upload.wikimedia.org/wikipedia/commons/4/4d/Northern_blot_diagram.png)
The northern blot, or RNA blot,
[Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates.] is a technique used in
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
research to study
gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
by detection of
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
(or isolated
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
) in a sample.
[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. 238:277–279.]
With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression rates during differentiation and
morphogenesis, as well as in abnormal or diseased conditions.
Northern blotting involves the use of
electrophoresis
Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fie ...
to separate RNA samples by size, and detection with a
hybridization probe complementary to part of or the entire target sequence. Strictly speaking, the term 'northern blot' refers specifically 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.
[Trayhurn, P. (1996) Northern Blotting. Pro. Nutrition Soc. 55:583–589.] The northern blot technique was developed in 1977 by James Alwine,
David Kemp, and George Stark at
Stanford University
Stanford University, officially Leland Stanford Junior University, is a private research university in Stanford, California. The campus occupies , among the largest in the United States, and enrolls over 17,000 students. Stanford is consider ...
. Northern blotting takes its name from its similarity to the first blotting technique, the
Southern blot
A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detecti ...
, named for biologist
Edwin Southern
Sir Edwin Mellor Southern (born 7 June 1938) is an English Lasker Award-winning molecular biologist, Emeritus Professor of Biochemistry at the University of Oxford and a fellow of Trinity College, Oxford. He is most widely known for the inventio ...
.
[Alberts, B., Johnson, A., Lewis, J. Raff, M., Roberts, K., Walter, P. 2008. Molecular Biology of the Cell, 5th ed. Garland Science, Taylor & Francis Group, NY, pp 538–539.] The major difference is that RNA, rather than
DNA, is analyzed in the northern blot.
Procedure
A general blotting procedure
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
Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically a messenger RNA (mRNA). The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In euk ...
.
RNA samples are then separated by gel electrophoresis. Since the gels 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. 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. The transfer buffer used for the blotting usually contains
formamide
Formamide is an amide derived from formic acid. It is a colorless liquid which is miscible with water and has an ammonia-like odor. It is chemical feedstock for the manufacture of sulfa drugs and other pharmaceuticals, herbicides and pesticide ...
because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating the need for high temperatures, which could cause RNA degradation.
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 affect the efficiency and specificity of hybridization include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition.
The membrane is washed to ensure that the probe has bound specifically and to prevent background signals from arising. The hybrid signals are then detected by X-ray film and can be quantified by
densitometry
Densitometry is the quantitative measurement of optical density in light-sensitive materials, such as photographic paper or photographic film, due to exposure to light.
Overview
Optical density is a result of the darkness of a developed picture ...
. 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
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
.
Gels
The RNA samples are most commonly separated on
agarose gels containing
formaldehyde
Formaldehyde ( , ) (systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section F ...
as a denaturing agent for the RNA to limit secondary structure.
The gels can be stained with
ethidium bromide
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It i ...
(EtBr) and viewed under UV light to observe the quality and quantity of RNA before blotting.
Polyacrylamide
Polyacrylamide (abbreviated as PAM) is a polymer with the formula (-CH2CHCONH2-). It has a linear-chain structure. PAM is highly water-absorbent, forming a soft gel when hydrated. In 2008, an estimated 750,000,000 kg were produced, mainly fo ...
gel electrophoresis with
urea
Urea, also known as carbamide, is an organic compound with chemical formula . This amide has two amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid.
Urea serves an important r ...
can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs.
[Valoczi, A., Hornyik, C., Varga, N., Burgyan, J., Kauppinen, S., Havelda, Z. (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nuc. Acids Research. 32: e175.] An RNA ladder is often run alongside 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.
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 to twice the intensity of the smaller.
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 (riboprobes) that are transcribed in vitro are able to withstand more rigorous washing steps preventing some of the background noise.
Commonly cDNA is created with labelled primers for the RNA sequence of interest to act as the probe in the northern blot.
[Liang, P. Pardee, A. B. (1995) Recent advances in differential display. Current Opinion Immunol. 7: 274–280.] The probes must be labelled either with radioactive isotopes (
32P) or with
chemiluminescence
Chemiluminescence (also chemoluminescence) is the emission of light (luminescence) as the result of a chemical reaction. There may also be limited emission of heat. Given reactants A and B, with an excited intermediate ◊,
: + -> lozenge -> ...
in which
alkaline phosphatase
The enzyme alkaline phosphatase (EC 3.1.3.1, alkaline phosphomonoesterase; phosphomonoesterase; glycerophosphatase; alkaline phosphohydrolase; alkaline phenyl phosphatase; orthophosphoric-monoester phosphohydrolase (alkaline optimum), systematic ...
or
horseradish peroxidase
The enzyme horseradish peroxidase (HRP), found in the roots of horseradish, is used extensively in biochemistry applications. It is a metalloenzyme with many isoforms, of which the most studied type is C. It catalyzes the oxidation of various or ...
(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
Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', bor ...
) for which the ligand (e.g.,
avidin
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians and deposited in the whites of their eggs. Dimeric members of the avidin family are also found in some bacteria. In chicken egg white, avidin ...
or
streptavidin
Streptavidin is a 66.0 (tetramer) kDa protein purified from the bacterium '' Streptomyces avidinii''. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation co ...
) 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.
Applications
Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment.
[Baldwin, D., Crane, V., Rice, D. (1999) A comparison of gel-based, nylon filter and microarray techniques to detect differential RNA expression in plants. Current Opinion in Plant Biol. 2: 96–103.] 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 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.
Advantages and disadvantages
Analysis of gene expression can be done by several different methods including RT-PCR, RNase protection assays, microarrays,
RNA-Seq
RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...
, serial analysis of gene expression (SAGE), as well as northern blotting.
Microarrays 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 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).
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.
Compared to RT-PCR, northern blotting has a low sensitivity, but it also has a high specificity, which is important to reduce false positive results.
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.
For northern blotting for the detection of
acetylcholinesterase
Acetylcholinesterase (HGNC symbol ACHE; EC 3.1.1.7; systematic name acetylcholine acetylhydrolase), also known as AChE, AChase or acetylhydrolase, is the primary cholinesterase in the body. It is an enzyme
Enzymes () are proteins that a ...
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
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.
[Kreft, K., Kreft, S., Komel, R., Grubič, Z. (2000). Nonradioactive northern blotting for the determination of acetylcholinesterase mRNA. Pflügers Arch – Eur J Physiol, 439:R66-R67]
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 affixed 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 microarray
A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to ...
s 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 affixed 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 profiling, in which many (possibly all) of the genes in an organism may have their expression monitored.
See also
*
Western blot
The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...
*
Eastern blot
The eastern blot, or eastern blotting, is a biochemical technique used to analyze protein post-translational modifications including the addition of lipids, phosphates, and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thu ...
*
Northwestern blot The northwestern blot, also known as the northwestern assay, is a hybrid analytical technique of the western blot and the northern blot, and is used in molecular biology to detect interactions between RNA and proteins. A related technique, the west ...
*
Far-eastern blot The far-eastern blot, or far-eastern blotting, is a technique for the analysis of lipids separated by high-performance thin layer chromatography ( HPTLC). When executing the technique, lipids are transferred from HPTLC plates to a PVDF membrane for ...
*
Far-western blot
The far-western blot, or far-western blotting, is a molecular biological method based on the technique of western blot to detect protein-protein interaction ''in vitro''. Whereas western blot uses an antibody probe to detect a protein of interest, ...
*
Differential display Differential display (also referred to as DDRT-PCR or DD-PCR) is a laboratory technique that allows a researcher to compare and identify changes in gene expression at the mRNA level between two or more eukaryotic cell samples. It was the most commo ...
References
External links
OpenWetWare
{{molecular genetics methods
Molecular biology techniques