Northern Blot- Definition, Principle, Steps, Results, Applications
RNA is a type of nucleic acid that carries the genetic information from DNA to the protein synthesis machinery in the cell. RNA molecules can have different functions and structures, such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and microRNA (miRNA). The expression of RNA can vary depending on the cell type, tissue, developmental stage, and environmental conditions. Therefore, studying RNA can provide insights into the regulation of gene expression and the molecular mechanisms of various biological processes and diseases.
However, analyzing RNA can be challenging because RNA molecules are often present in low amounts and are easily degraded by enzymes called RNases. Moreover, RNA molecules can have different lengths and sequences, which makes it difficult to identify and separate them from a complex mixture. To overcome these challenges, molecular biologists have developed a technique called Northern blot, which allows the detection and quantification of specific RNA molecules among a mixture of RNA.
Northern blot is named after its similarity to another technique called Southern blot, which is used to analyze DNA. Northern blot involves four main steps:
- Separation of RNA on a denaturing gel by electrophoresis
- Transfer of RNA from gel to a nylon membrane by capillary action
- Immobilization of RNA on the membrane by UV cross-linking or baking
- Hybridization of the membrane with a labeled probe that is complementary to the target RNA sequence
The labeled probe can be detected by various methods, such as autoradiography, chemiluminescence, or fluorescence. The intensity and position of the signal on the membrane indicate the amount and size of the target RNA molecule in the sample.
Northern blot is a powerful technique that can be used for various purposes, such as:
- Identifying and characterizing different types of RNA molecules, such as mRNA, tRNA, rRNA, and miRNA
- Measuring the expression levels of specific genes in different tissues or cells
- Comparing the expression patterns of genes under different conditions or treatments
- Detecting mutations, deletions, insertions, or splicing variants in RNA sequences
- Studying the structure and function of RNA molecules
In this article, we will explain the principle, equipment, materials, steps, results, applications, and limitations of Northern blot in detail. We will also provide some examples and tips for performing this technique successfully.
The principle of the northern blot is based on the transfer of RNA molecules from a gel to a membrane, followed by the detection of specific RNA sequences using complementary probes.
The first step in northern blotting is to separate the RNA samples by size using gel electrophoresis. Since RNA molecules are single-stranded and can form secondary structures by base pairing, the electrophoresis is performed under denaturing conditions to ensure that the RNA molecules are fully extended and separated according to their length.
The second step is to transfer the separated RNA molecules from the gel to a nylon membrane. This can be done by capillary action or vacuum suction, using a buffer solution that facilitates the binding of RNA to the membrane. The nylon membrane is preferred over the nitrocellulose membrane because it has a higher affinity for RNA and can withstand harsh conditions.
The third step is to immobilize the RNA molecules on the membrane by cross-linking them with UV light or heat. This prevents the RNA from being washed off or degraded during subsequent steps.
The fourth step is to hybridize the membrane with a labeled probe that is complementary to the target RNA sequence. The probe can be either DNA or RNA, and it can be labeled with a radioactive or non-radioactive marker, such as biotin or digoxigenin. The probe anneals to the target RNA sequence on the membrane, forming a double-stranded hybrid. The hybridization conditions, such as temperature, salt concentration, and probe concentration, affect the specificity and sensitivity of the detection.
The fifth step is to wash the membrane with a solution that removes any excess or non-specifically bound probe. The washing conditions, such as temperature and stringency, also affect the specificity and sensitivity of the detection.
The final step is to visualize and quantify the hybridized probe on the membrane using autoradiography or chemiluminescence. The intensity of the signal corresponds to the amount of target RNA present in the sample. The size of the target RNA can be estimated by comparing it with a standard RNA ladder run on the same gel.
Northern blotting allows the analysis of gene expression by detecting and measuring specific RNA molecules in a complex mixture. It can also provide information about the structure and processing of RNA molecules, such as splicing, editing, and polyadenylation.
To perform a Northern blot, you will need the following equipment and materials:
- Agarose gel cast: A device to pour and set the agarose gel for electrophoresis.
- Power supply: A device to provide an electric current to run the electrophoresis.
- Microwave: A device to heat and melt the agarose gel solution.
- Centrifuge: A device to spin and separate the RNA samples from debris or contaminants.
- Heating block: A device to heat and denature the RNA samples before loading them on the gel.
- UV crosslinker: A device to expose the nylon membrane with transferred RNA to UV rays and immobilize the RNA on the membrane.
- Hybridization oven: A device to incubate the membrane with the labeled probe at a controlled temperature and agitation.
- Hybridization vessels: Containers to hold the membrane and the hybridization solution during incubation.
- Vials: Containers to hold the probe and other reagents.
- Forceps: Tools to handle the gel and the membrane.
- Pipettes: Tools to transfer liquids such as samples, buffers, and probes.
- Glass tubes: Containers to hold the samples during heating or centrifugation.
You will also need the following reagents and materials:
- Agarose gel: A polysaccharide matrix that forms pores for RNA separation by size.
- Sodium citrate: A buffer component that stabilizes the pH and prevents RNA degradation.
- Ethylenediaminetetraacetic acid disodium salt dehydrates (EDTA): A chelating agent that binds metal ions and inhibits RNase activity.
- NaOH: A strong base that denatures double-stranded RNA into single-stranded RNA.
- HCl: A strong acid that neutralizes the NaOH after denaturation.
- Formaldehyde: A denaturing agent that prevents RNA secondary structures and improves separation by size.
- Glycerol: A viscous liquid that increases the density of the RNA samples and helps them sink into the wells of the gel.
- Ethidium bromide: A fluorescent dye that intercalates with RNA and allows visualization under UV light.
- Bromophenol Blue: A tracking dye that indicates the progress of electrophoresis and helps estimate the size of RNA fragments.
- RNA ladder: A mixture of known RNA fragments that serve as a reference for size determination of unknown RNA samples.
- MgCl2: A cofactor for some enzymes involved in probe generation or labeling.
- NaCl: A salt that affects the stringency of hybridization and washing steps.
- Polyvinylpyrrolidone (PVP): A blocking agent that reduces non-specific binding of probes to the membrane or other molecules.
- Bovine Serum Albumin (BSA): Another blocking agent that reduces the non-specific binding of probes to the membrane or other molecules.
- SDS: A detergent that solubilizes proteins and lipids and reduces background noise.
- NaH2PO4: A buffer component that stabilizes the pH and prevents RNA degradation.
- Tris-HCl: Another buffer component that stabilizes the pH and prevents RNA degradation.
- Triton-X: Another detergent that solubilizes proteins and lipids and reduces background noise.
- DTT: A reducing agent that breaks disulfide bonds and prevents oxidation of RNA or probes.
- Taq buffer: A buffer component that provides optimal conditions for Taq polymerase activity in probe generation or labeling.
- Taq polymerase: An enzyme that synthesizes DNA from a template using dNTPs in probe generation or labeling.
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The Northern Blot procedure can be divided into four main steps: separation of RNA on a denaturing gel, transfer of RNA from gel to a nylon membrane, immobilization, and hybridization. Each step requires specific equipment and materials, as well as careful handling of the RNA samples to avoid degradation and contamination. The following is a brief description of each step with some tips and precautions.
a. Separation of RNA on a denaturing gel
The first step of the Northern Blot procedure is to separate the RNA samples according to their size by gel electrophoresis. This step requires a denaturing agarose gel that contains formaldehyde, which prevents the formation of secondary structures by RNA molecules. The gel is prepared by dissolving agarose in water and adding formaldehyde and sodium citrate buffer. The gel is then poured into a cast with a comb that forms wells for loading the samples.
The RNA samples are mixed with an equal volume of RNA loading buffer that contains glycerol, ethidium bromide, bromophenol blue, and formaldehyde. The loading buffer helps to visualize the samples on the gel and to denature the RNA molecules. The samples are then heated at 65°C for about 15 minutes to ensure complete denaturation.
The samples are loaded onto the wells of the gel along with an RNA ladder that serves as a size marker. The gel is then run at a constant voltage of 125V for about 3 hours in an electrophoresis tank filled with sodium citrate buffer. The electrophoresis separates the RNA molecules according to their size, with smaller molecules migrating faster than larger ones.
The gel is then removed from the tank and rinsed with water. The RNA bands can be visualized under UV light by using ethidium bromide as a fluorescent dye that intercalates with nucleic acids. Alternatively, the gel can be stained with other dyes such as SYBR Green or methylene blue.
b. Transfer of RNA from gel to a nylon membrane
The second step of the Northern Blot procedure is to transfer the separated RNA molecules from the gel to a nylon membrane that can bind them tightly and stably. This step requires a capillary transfer system that consists of a glass dish, a sponge, filter papers, a nylon membrane, and a glass plate.
The nylon membrane is cut slightly larger than the size of the gel and soaked in distilled water for about 5 minutes. The membrane is then placed on top of the gel and aligned carefully to avoid air bubbles. The membrane should be handled with gloves and forceps to avoid contamination and damage.
The gel-membrane sandwich is then placed on top of several layers of wet filter papers that are placed on top of a soaked sponge in a glass dish filled with sodium citrate buffer (SSC). More layers of wet filter papers are placed on top of the membrane and covered with a glass plate that provides some weight and pressure.
The capillary action of the SSC buffer draws the RNA molecules from the gel to the membrane through the filter papers. The transfer process takes about 12 hours or overnight to ensure a complete transfer.
The third step of the Northern Blot procedure is to immobilize the transferred RNA molecules onto the nylon membrane by using heat or UV light. This step prevents the loss or displacement of the RNA molecules during subsequent hybridization and washing steps.
The membrane is removed from the transfer system and rinsed briefly with SSC buffer. The membrane is then placed between two pieces of filter paper and baked in a vacuum oven at 80°C for 2 hours. Alternatively, the membrane can be wrapped in a UV transparent plastic wrap and irradiated for an appropriate time on a UV crosslinker.
The immobilization step ensures that the RNA molecules are covalently attached to the nylon membrane and can withstand high temperatures and harsh conditions during hybridization.
The fourth and final step of the Northern Blot procedure is to hybridize the immobilized RNA molecules with a labeled probe that is complementary to the target RNA sequence of interest. This step allows the detection and identification of specific RNA molecules on the membrane.
The probe can be either DNA or RNA, single-stranded or double-stranded, and labeled with radioactive or non-radioactive methods such as biotin, digoxigenin, or fluorescent dyes. The probe should be purified from unincorporated nucleotides and denatured if double-stranded before hybridization.
The membrane is pre-hybridized in a hybridization tube with a formaldehyde solution that contains sodium chloride, sodium phosphate buffer, polyvinylpyrrolidone, bovine serum albumin, sodium dodecyl sulfate (SDS), Triton-X and dithiothreitol (DTT). The pre-hybridization step reduces the non-specific binding of the probe to the membrane and improves the signal-to-noise ratio.
The pre-hybridized membrane is then incubated with the labeled probe in a hybridization oven at 42°C for several hours or overnight. The hybridization temperature depends on the type and length of the probe and should be optimized for each experiment.
The hybridized membrane is then washed with wash solutions that contain sodium chloride, sodium phosphate buffer, and SDS at different temperatures and stringencies. The wash steps remove the excess probe and background signals from the membrane.
The hybridized membrane is then exposed to autoradiography film or detected by other methods depending on the type of label used for the probe. The presence of bands on the film or signal on the detector indicates positive hybridization between the probe and target RNA molecules on the membrane.
The RNA bands are observed under radiography in the form of bands. The distance of the bands from the markers can be used to determine the length and semi-quantification of the RNA fragments. The intensity of the bands indicates the relative abundance of the target RNA in the sample. The more intense the band, the more RNA is present in the sample. The size of the band can also indicate the presence of alternative splicing or post-transcriptional modifications.
To compare the expression levels of different RNA samples, it is important to normalize the data by using a housekeeping gene as an internal control. A housekeeping gene is a gene that is expressed at a constant level in all cells and tissues, regardless of environmental or physiological conditions. Examples of housekeeping genes are GAPDH, β-actin, and 18S rRNA. By hybridizing the same membrane with a probe for a housekeeping gene, one can obtain a reference band that can be used to correct for variations in loading and transfer efficiency. The ratio of the target band intensity to the reference band intensity can then be calculated and compared across different samples.
To verify the specificity of the hybridization signal, one can also perform a control experiment by using a probe that is not complementary to the target RNA. This probe should not produce any signal on the membrane, indicating that there is no cross-hybridization or background noise. Alternatively, one can use a probe that is complementary to a different region of the target RNA and compare the results with the original probe.
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If there is no signal or a very weak signal on the membrane, it could indicate that the RNA sample was degraded, insufficient, or poorly transferred. To check for these problems, one can stain the gel or membrane with ethidium bromide or methylene blue and visualize the ribosomal RNA bands. These bands should be clear and sharp, indicating intact and high-quality RNA.
If there are multiple bands or unexpected sizes on the membrane, it could indicate that the RNA sample contains different isoforms or variants of the target RNA. These could result from alternative splicing, alternative polyadenylation, intron retention, or editing events. To confirm these possibilities, one can sequence the bands or perform RT-PCR with specific primers.
Northern blotting is a powerful technique that can be used for various purposes in molecular biology research. Some of the applications of northern blotting are:
- Identification and separation of RNA fragments collected from different biological sources. Northern blotting can be used to compare the RNA profiles of different tissues, cell types, organisms, or experimental conditions.
- Detection and quantification of specific mRNAs from a complex RNA mixture. Northern blotting can be used to measure the expression levels of particular genes by hybridizing a specific probe to the target mRNA on the membrane. The intensity of the signal can be used to estimate the abundance and size of the mRNA.
- Gene expression studies such as determining when and where a particular gene is expressed. Northern blotting can be used to analyze the temporal and spatial patterns of gene expression during development, differentiation, morphogenesis, stress response, disease progression, or treatment.
- Detection of transcriptional regulation of DNA fragments that are used as probes in Southern blotting. Northern blotting can be used as a sensitive test to determine if a DNA fragment is transcribed into RNA in a given sample. This can help to identify promoters, enhancers, or other regulatory elements that control gene expression.
- Detection of RNA processing events such as splicing, editing, cleavage, or polyadenylation. Northern blotting can be used to examine the structure and diversity of RNA transcripts generated from a single gene. This can reveal alternative splicing patterns, RNA editing sites, or poly (A) tail lengths that may affect the function or stability of the RNA.
- Detection of viral or bacterial RNAs that play important roles in infection or pathogenesis. Northern blotting can be used to identify and quantify viral or bacterial RNAs in infected cells or tissues. This can help to diagnose infections, monitor viral load, or study viral replication mechanisms.
- Detection of non-coding RNAs such as microRNAs, small interfering RNAs, or long non-coding RNAs that regulate gene expression at various levels. Northern blotting can be used to analyze the expression and function of these non-coding RNAs that are involved in various biological processes and diseases.
Northern blotting is a powerful technique for analyzing specific RNA in a complex mixture, but it also has some limitations that should be considered. Some of the limitations are:
- Low sensitivity: Northern blotting has a lower sensitivity than other techniques, such as RT-PCR and nuclease protection assays, which can detect RNA at much lower concentrations. This means that Northern blotting may not be able to detect low-abundance RNA or RNA that is degraded or unstable. The sensitivity of Northern blotting can be improved by using more sensitive probes, such as digoxigenin-labeled probes, or by using chemiluminescence or fluorescence detection methods instead of autoradiography.
- Large sample requirement: Northern blotting requires a large amount of sample RNA, usually in the range of 10-20 µg per lane. This can be a problem when the RNA source is limited or precious, such as in tissue biopsies or clinical samples. The sampling requirement can be reduced by using mini-gels or microarrays, but this may compromise the resolution and accuracy of the technique.
- Time-consuming and complex procedure: Northern blotting is a time-consuming and complex procedure that involves multiple steps, such as RNA extraction, gel electrophoresis, transfer, immobilization, hybridization, washing, and detection. Each step requires careful optimization and quality control to ensure reliable results. The procedure can take several days to complete, especially when multiple probes are used. The procedure can be simplified by using precast gels, prehybridized membranes, or commercial kits.
- Non-specific hybridization: Northern blotting relies on the specificity of hybridization between the probe and the target RNA to detect the desired RNA. However, non-specific hybridization can occur due to the presence of homologous sequences in the sample or the probe or due to suboptimal hybridization conditions. Non-specific hybridization can result in false-positive or false-negative signals or interfere with the quantification of the target RNA. Non-specific hybridization can be minimized by using high-quality probes and RNA samples, optimizing the hybridization temperature and stringency, and using appropriate blocking and washing solutions.
These limitations should be taken into account when choosing Northern blotting as a technique for RNA analysis. However, despite these limitations, Northern blotting remains a valuable and widely used technique for studying gene expression and RNA structure.
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