Northern blot
Overview
 
The northern blot is a technique used in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 research to study gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...

 by detection of RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

 (or isolated mRNA) in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis
Morphogenesis
Morphogenesis , is the biological process that causes an organism to develop its shape...

, as well as abnormal or diseased conditions. Northern blotting involves the use of electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...

 to separate RNA samples by size and detection with a hybridization probe
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...

 complementary to part of or the entire target sequence.
Encyclopedia
The northern blot is a technique used in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 research to study gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...

 by detection of RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

 (or isolated mRNA) in a sample.
With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis
Morphogenesis
Morphogenesis , is the biological process that causes an organism to develop its shape...

, as well as abnormal or diseased conditions. Northern blotting involves the use of electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...

 to separate RNA samples by size and detection with a hybridization probe
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...

 complementary to part of or the entire target sequence. The term 'northern blot' actually 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. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot
Southern blot
A Southern blot is a method routinely 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 detection by probe hybridization. The method is named...

, named for biologist Edwin Southern
Edwin Southern
Sir Edwin Mellor Southern, FRS is an English 2005 Lasker Award-winning molecular biologist. His award was for the invention of the Southern blot, now a common laboratory procedure, when he was working at the University of Edinburgh....

. The major difference is that RNA, rather than DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

, 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
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....

 to isolate only those RNAs with a poly(A) tail. 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
Formamide, also known as methanamide, is an amide derived from formic acid. It is a clear liquid which is miscible with water and has an ammonia-like odor. It is used primarily for manufacturing sulfa drugs and synthesizing vitamins and as a softener for paper and fiber...

 because it lowers the annealing temperature of the probe-RNA interaction, thus preventing RNA degradation by high temperatures. 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 avoid background signals from arising. The hybrid signals are then detected by X-ray film and can be quantified by densitometry
Densitometry
Densitometry is the quantitative measurement of optical density in light-sensitive materials, such as photographic paper or film, due to exposure to light...

. 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.

Gels

The RNA samples are most commonly separated on agarose gels containing formaldehyde
Formaldehyde
Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...

 as a denaturing agent for the RNA to limit secondary structure. The gels can be stained with ethidium bromide
Ethidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...

 (EtBr) and viewed under UV light to observe the quality and quantity of RNA before blotting. Polyacrylamide
Polyacrylamide
Polyacrylamide is a polymer formed from acrylamide subunits. It can be synthesized as a simple linear-chain structure or cross-linked, typically using N,N-methylenebisacrylamide. Polyacrylamide is not toxic...

 gel electrophoeresis with urea
Urea
Urea or carbamide is an organic compound with the chemical formula CO2. The molecule has two —NH2 groups joined by a carbonyl functional group....

 can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs. 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 will 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. The probes need to be labelled either with radioactive isotopes (32P) or with chemiluminescence in which alkaline phosphatase
Alkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...

 or horseradish peroxidase
Horseradish peroxidase
The enzyme horseradish peroxidase , found in horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.-Applications:...

 breakdown 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
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...

) for which the antibody (e.g. avidin
Avidin
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein...

 or streptavidin
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...

) is attached to the enzyme. 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. 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.

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 tissue. The results of a search provide the blot ID, species, tissue, gene, expression level, blot image (if available), and links to the publication that the work originated from. This new database provides sharing of information between members of the science community that was not previously seen in northern blotting as it was in sequence analysis, genome determination, protein structure, etc.

Advantages and disadvantages

Analysis of gene expression can be done by several different methods including RT-PCR, RNase protection assays, microarrays, 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
Diethylpyrocarbonate
Diethylpyrocarbonate , also called diethyl dicarbonate , diethyl oxydiformate, ethoxyformic anhydride, or pyrocarbonic acid diethyl ester, is used in the laboratory to inactivate the RNase enzymes from water and other laboratory utensils. It inactivates the RNases by the covalent modifications of...

). 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.

Reverse northern blot

A variant of the procedure known as the reverse northern blot is occasionally used. 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
DNA microarray
A DNA microarray 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 genotype multiple regions of a genome...

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
Expression profiling
In the field of molecular biology, gene expression profiling is the measurement of the activity of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between cells that are actively dividing, or show how the cells react to a...

, in which many (possibly all) of the genes in an organism may have their expression monitored.

See also

  • Western blot
    Western blot
    The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...

  • Far-Eastern blotting
  • Far-western blotting
    Far-western blotting
    Far-Western blotting is a molecular biological method which is based on the technique of Western blotting. While usual Western blotting uses an antibody to detect a protein of interest, far-Western blotting uses a non-antibody protein, which can bind the protein of interest...

  • Eastern blotting
    Eastern blotting
    Eastern blotting is a biochemical technique used to analyze protein post translational modifications such as lipids and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thus, Eastern blotting can be considered an extension of the biochemical technique of Western blotting...

  • Differential display
    Differential display
    Differential display is the technique where a researcher can compare and identify changes in gene expression at the mRNA level between any pair of eukaryotic cell samples. The assay may be extended to more than one pair, if needed...

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