Northern blot

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Flow diagram outlining the general procedure for RNA detection by northern blotting. Northern blot diagram.png
Flow diagram outlining the general procedure for RNA detection by northern blotting.

The northern blot, or RNA blot, [1] is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. [2] [3]

Contents

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. [4] 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. 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. [5] The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. [6] Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern. [2] The major difference is that RNA, rather than DNA, is analyzed in the northern blot. [7]

Procedure

A general blotting procedure [5] 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. [8] [9] 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.

Capillary blotting system setup for the transfer of RNA from an electrophoresis gel to a blotting membrane. Capillary blot setup.svg
Capillary blotting system setup for the transfer of RNA from an electrophoresis gel to a blotting membrane.

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 because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating the need for high temperatures, which could cause RNA degradation. [10] 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. [11] 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. 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. [11]

Gels

RNA run on a formaldehyde agarose gel to highlight the 28S (top band) and 18S (lower band) ribosomal subunits. RNA agarose gel.svg
RNA run on a formaldehyde agarose gel to highlight the 28S (top band) and 18S (lower band) ribosomal subunits.

The RNA samples are most commonly separated on agarose gels containing formaldehyde as a denaturing agent for the RNA to limit secondary structure. [11] [12] The gels can be stained with ethidium bromide (EtBr) and viewed under UV light to observe the quality and quantity of RNA before blotting. [11] Polyacrylamide gel electrophoresis with urea can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs. [13] 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. [11] 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. [11] [14]

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. [5] RNA probes (riboprobes) that are transcribed in vitro are able to withstand more rigorous washing steps preventing some of the background noise. [11] Commonly cDNA is created with labelled primers for the RNA sequence of interest to act as the probe in the northern blot. [15] The probes must be labelled either with radioactive isotopes (32P) or with chemiluminescence in which alkaline phosphatase or horseradish peroxidase (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 ligand (e.g. biotin) for which the ligand (e.g., avidin or streptavidin) is attached to the enzyme (e.g. HRP). [11] 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. [16] The same membrane can be probed up to five times without a significant loss of the target RNA. [10]

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. [9] [15] [17] The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue, [11] as well as the gene expression in the rejection of transplanted organs. [18] 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. [18] 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. [8] [14] 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. [2]

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. [4] [5] 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. [19] 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. [17] [19]

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). [5] 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. [11] 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. [11]

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. [11]

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. [20]

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 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 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

Related Research Articles

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 physical structure of biological macromolecules is known as molecular biology.

In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, populations, or species or to pinpoint the locations of genes within a sequence. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.

<span class="mw-page-title-main">Southern blot</span> DNA analysis technique

Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample is digested with restriction enzymes, and the resulting DNA fragments are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.

<span class="mw-page-title-main">Gene expression</span> Conversion of a genes sequence into a mature gene product or products

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. These products are often proteins, but in non-protein-coding genes such as transfer RNA (tRNA) and small nuclear RNA (snRNA), the product is a functional non-coding RNA. Gene expression is summarized in the central dogma of molecular biology first formulated by Francis Crick in 1958, further developed in his 1970 article, and expanded by the subsequent discoveries of reverse transcription and RNA replication.

<span class="mw-page-title-main">Western blot</span> Analytical technique used in molecular biology

The western blot, 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 detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination.

<span class="mw-page-title-main">Reverse transcription polymerase chain reaction</span> Laboratory technique to multiply an RNA sample for study

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.

<span class="mw-page-title-main">DNA microarray</span> Collection of microscopic DNA spots attached to a solid surface

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. Each DNA spot contains picomoles of a specific DNA sequence, known as probes. These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. The original nucleic acid arrays were macro arrays approximately 9 cm × 12 cm and the first computerized image based analysis was published in 1981. It was invented by Patrick O. Brown. An example of its application is in SNPs arrays for polymorphisms in cardiovascular diseases, cancer, pathogens and GWAS analysis. It is also used for the identification of structural variations and the measurement of gene expression.

<span class="mw-page-title-main">Blot (biology)</span>

A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA onto a carrier. In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining, autoradiographic visualization of radiolabelled molecules, or specific labelling of some proteins or nucleic acids. The latter is done with antibodies or hybridization probes that bind only to some molecules of the blot and have an enzyme joined to them. After proper washing, this enzymatic activity is visualized by incubation with proper reactive, rendering either a colored deposit on the blot or a chemiluminescent reaction which is registered by photographic film.

This is a list of topics in molecular biology. See also index of biochemistry articles.

In molecular biology, a hybridization probe(HP) is a fragment of DNA or RNA of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of nucleotide sequences in analyzed RNA or DNA that are complementary to the sequence in the probe. The labeled probe is first denatured (by heating or under alkaline conditions such as exposure to sodium hydroxide) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ.

Fluorescence <i>in situ</i> hybridization Genetic testing technique

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

<i>In situ</i> hybridization

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand to localize a specific DNA or RNA sequence in a portion or section of tissue or if the tissue is small enough, in the entire tissue, in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

<span class="mw-page-title-main">Southwestern blot</span> Molecular biology technique

The southwestern blot, is a lab technique that involves identifying as well as characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes. Determination of molecular weight of proteins binding to DNA is also made possible by the technique. The name originates from a combination of ideas underlying Southern blotting and Western blotting techniques of which they detect DNA and protein respectively. Similar to other types of blotting, proteins are separated by SDS-PAGE and are subsequently transferred to nitrocellulose membranes. Thereafter southwestern blotting begins to vary with regards to procedure as since the first blotting’s, many more have been proposed and discovered with goals of enhancing results. Former protocols were hampered by the need for large amounts of proteins and their susceptibility to degradation while being isolated.

<span class="mw-page-title-main">Real-time polymerase chain reaction</span> Laboratory technique of molecular biology

A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.

<span class="mw-page-title-main">RNA spike-in</span>

An RNA spike-in is an RNA transcript of known sequence and quantity used to calibrate measurements in RNA hybridization assays, such as DNA microarray experiments, RT-qPCR, and RNA-Seq.

An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler Dot blot assay. It is a common tool used in genetic testing, forensics, and Molecular Biology research.

<span class="mw-page-title-main">MAGIChip</span>

MAGIChips, also known as "microarrays of gel-immobilized compounds on a chip" or "three-dimensional DNA microarrays", are devices for molecular hybridization produced by immobilizing oligonucleotides, DNA, enzymes, antibodies, and other compounds on a photopolymerized micromatrix of polyacrylamide gel pads of 100x100x20µm or smaller size. This technology is used for analysis of nucleic acid hybridization, specific binding of DNA, and low-molecular weight compounds with proteins, and protein-protein interactions.

The reverse northern blot is a method by which gene expression patterns may be analyzed by comparing isolated RNA molecules from a tester sample to samples in a control cDNA library. It is a variant of the northern blot in which the nucleic acid immobilized on a membrane is a collection of isolated DNA fragments rather than RNA, and the probe is RNA extracted from a tissue and radioactively labelled. A reverse northern blot can be used to profile expression levels of particular sets of RNA sequences in a tissue or to determine presence of a particular RNA sequence in a sample. Although DNA Microarrays and newer next-generation techniques have generally supplanted reverse northern blotting, it is still utilized today and provides a relatively cheap and easy means of defining expression of large sets of genes.

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 western blot, is used to detect a protein of interest that involves transferring proteins that are separated by gel electrophoresis onto a nitrocellulose membrane. A colored precipitate clusters along the band on the membrane containing a particular target protein. A northern blot is a similar analytical technique that, instead of detecting a protein of interest, is used to study gene expression by detection of RNA on a similar membrane. The northwestern blot combines the two techniques, and specifically involves the identification of labeled RNA that interact with proteins that are immobilized on a similar nitrocellulose membrane.

Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst non-coding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell. Transcriptomics technologies provide a broad account of which cellular processes are active and which are dormant. A major challenge in molecular biology is to understand how a single genome gives rise to a variety of cells. Another is how gene expression is regulated.

References

  1. ↑ Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates.
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  5. 1 2 3 4 5 Trayhurn, P. (1996) Northern Blotting. Pro. Nutrition Soc. 55:583–589.
  6. ↑ Alwine JC, Kemp DJ, Stark GR (1977). "Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5350–4. Bibcode:1977PNAS...74.5350A. doi: 10.1073/pnas.74.12.5350 . PMC   431715 . PMID   414220.
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  12. ↑ 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): 321–333. doi: 10.1101/gad.11.3.321 . PMID   9030685.
  13. ↑ 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.
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  19. 1 2 Taniguchi, M.; Miura, K.; Iwao, H.; Yamanaka, S. (2001). "Quantitative Assessment of DNA Microarrays – Comparison with Northern Blot Analysis". Genomics. 71 (1): 34–39. doi:10.1006/geno.2000.6427. PMID   11161795.
  20. ↑ 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
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