Northern Blotting – Protocol, Principle, Application, Result

The northern blot, or RNA blot, is a technique utilized in molecular biology research to examine gene expression through the detection of RNA (or isolated mRNA) in a sample.

Using northern blotting, it is possible to observe cellular control over structure and function by determining gene expression rates during differentiation, morphogenesis, and disease.Northern blotting utilizes electrophoresis to size-separate RNA samples and a hybridization probe complementary to a portion or the entire target sequence for detection. The term ‘northern blot’ strictly refers to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. Nevertheless, the entire procedure is commonly known as northern blotting. The northern blot technique was devised in 1977 at Stanford University by James Alwine, David Kemp, and George Stark. Northern blotting is akin to the first blotting technique, the Southern blot, which was named after the biologist Edwin Southern.Northern blot analysis focuses on RNA instead of DNA.

What is Northern Blotting Technique?

Northern blotting is a laboratory technique used to detect and analyze specific RNA sequences within a complex mixture, providing information about RNA length and sequence variations.

• Northern blotting is a technique rooted in the principle of blotting, designed to analyze specific RNA sequences within a complex mixture. This method is a derivative of Southern blotting, initially devised for DNA sequence analysis. The distinction lies in the type of nucleic acid being detected: Northern blotting focuses on RNA sequences.
• In molecular biology, detecting particular nucleic acid sequences extracted from various biological samples is crucial. This necessity renders blotting techniques indispensable in the field. The Northern blotting method mirrors the Southern blotting process, with the key difference being the probes used for detection; Northern blotting employs probes to detect RNA sequences.
• The technique yields critical information about the length of RNA sequences and the presence of sequence variations. While primarily used for identifying RNA sequences, Northern blotting also serves in quantifying RNA sequences. Since its inception, the method has undergone several modifications to facilitate the analysis of mRNAs, pre-mRNAs, and short RNAs.
• Historically, Northern blotting was the primary technique for RNA fragment analysis. However, advancements have introduced more convenient and cost-effective methods, such as RT-PCR, which have gradually supplanted Northern blotting in many applications.

Aim of Northern Blotting

• To learn the technique of Northern Blotting for the detection of a specific RNA fragment in a sample

Northern Blotting principle

• The principle of Northern blotting aligns with other blotting techniques, relying on the transfer of biomolecules from a gel to a membrane. Initially, RNA samples are separated by size through gel electrophoresis. Due to the single-stranded nature of RNA, these molecules can form secondary structures via intermolecular base pairing. Thus, electrophoretic separation is conducted under denaturing conditions to prevent secondary structure formation.
• Following separation, the RNA fragments are transferred to a nylon membrane, as nitrocellulose membranes are less effective at binding RNA. Once transferred, the RNA fragments are immobilized onto the membrane using fixing agents. Detection of these RNA fragments involves adding a labeled probe that is complementary to the target RNA sequences on the membrane.
• The process of hybridization, wherein the probe binds specifically to its complementary RNA sequence, underpins the detection mechanism. This specificity allows for accurate identification of RNA segments. Additionally, Northern blotting, which utilizes size-dependent separation of RNA fragments, is instrumental in determining the sizes of transcripts.

Material Required for Northern Blotting

Equipment

1. Agarose Gel Cast: Utilized for preparing the gel in which RNA samples are separated by electrophoresis.
2. Power Supply: Provides the necessary voltage for gel electrophoresis to separate RNA fragments.
3. Microwave: Used to melt agarose for gel preparation.
4. Centrifuge: Assists in sample preparation by separating components at high speeds.
5. Heating Block: Maintains consistent temperatures for various reaction steps.
6. UV Crosslinker: Fixes RNA to the membrane by using ultraviolet light.
7. Hybridization Oven: Ensures optimal conditions for probe hybridization with the RNA on the membrane.
8. Hybridization Vessels: Holds membranes and probes during the hybridization process.
9. Vials: Used for preparing and storing various solutions.
10. Forceps: Essential for handling membranes without contamination.
11. Pipettes: Accurate liquid handling for transferring small volumes.
12. Glass Tubes: Containers for reactions and hybridizations.

Materials

1. Agarose Gel: The medium in which RNA samples are separated during electrophoresis.
2. Sodium Citrate: A buffering agent used in various preparation steps.
3. Ethylenediaminetetraacetic Acid Disodium Salt Dehydrate (EDTA): Chelates divalent metal ions, protecting RNA from degradation.
4. NaOH: Sodium hydroxide, used in denaturing RNA.
5. HCl: Hydrochloric acid, also involved in RNA denaturation.
6. Formaldehyde: A denaturing agent that prevents secondary structure formation in RNA.
7. Glycerol: Adds density to samples for easier loading into gel wells.
8. Ethidium Bromide: A fluorescent dye used to visualize RNA under UV light.
9. Bromophenol Blue: A tracking dye that helps monitor the progress of electrophoresis.
10. RNA Ladder: Size markers to estimate the length of RNA fragments.
11. MgCl2: Magnesium chloride, used in buffer solutions.
12. NaCl: Sodium chloride, another common buffer component.
13. Polyvinylpyrrolidone (PVP): Prevents non-specific binding in hybridization reactions.
14. Bovine Serum Albumin (BSA): Stabilizes enzymes and blocks non-specific sites.
15. Sodium Dodecyl Sulfate (SDS): A detergent that denatures proteins.
16. NaH2PO4: Sodium phosphate, a buffering agent.
17. Tris-HCl: Tris(hydroxymethyl)aminomethane hydrochloride, a buffering agent.
18. Triton-X: A non-ionic detergent used in various buffer solutions.
19. Dithiothreitol (DTT): A reducing agent that prevents RNA degradation.
20. Taq Buffer: Buffer solution used for stabilizing conditions in enzymatic reactions.
21. Taq Polymerase: An enzyme used in polymerase chain reactions for amplifying RNA.

Solutions & buffers Preparation

1. MOPS Buffer

MOPS (3-(N-morpholino)propanesulfonic acid) Buffer is crucial for maintaining a stable pH during the electrophoresis of RNA samples.

• 10× MOPS Buffer Preparation:
  • Components:
    • MOPS: 0.2 M (41.852 g)
    • Sodium acetate •3H2O: 80 mM (10.89 g)
    • EDTA: 10 mM (0.372 g)
  • Instructions:
    1. Dissolve the components in water.
    2. Adjust the pH to 7.0 with NaOH.
    3. Add water to make up to 1 liter.
    4. Store at room temperature, protected from light.

2. Denaturing RNA Gel

Denaturing RNA Gel is used to separate RNA molecules by size during electrophoresis.

• Preparation:
  • Components (for 150 ml):
    • MOPS buffer (10×): 15 ml
    • Agarose: 1.2% (w/v) (1.8 g)
    • H2O: 132 ml
  • Instructions:
    1. Microwave the mixture for 3-4 minutes to dissolve agarose.
    2. Cool until it can be held comfortably.
    3. Add 2.8 ml of formaldehyde.

3. Running Buffer

Running Buffer facilitates the movement of RNA through the gel during electrophoresis.

• Preparation:
  • Components:
    • MOPS buffer (10×): 100 ml
    • Formaldehyde: 7% (from 37% stock, 19 ml)
  • Instructions:
    1. Add water to make up to 1 liter.

4. RNA Loading Buffer

RNA Loading Buffer ensures proper sample loading and visualization during electrophoresis.

• Preparation:
  • Components (for 10 ml):
    • MOPS buffer (10×): 1 ml
    • Glycerol: 20% (2 ml)
    • Formaldehyde: 6.5% (1.76 ml)
    • Formamide: 50% (5 ml)
    • Ethidium Bromide: 10 μg/ml (100 μg)
    • Bromophenol Blue: 0.05% (w/v) (5 mg)
    • Xylene Cyanol: 0.05% (w/v) (5 mg)
  • Instructions:
    1. Add water to make up to 10 ml.

5. SSC Buffer

SSC (Saline-Sodium Citrate) Buffer is used in the hybridization and washing steps of the blotting process.

• 20× SSC Buffer Preparation:
  • Components:
    • NaCl: 3 M (175.3 g)
    • Sodium Citrate: 300 mM (88.2 g)
  • Instructions:
    1. Dissolve the components in water.
    2. Adjust the pH to 7.0 with HCl.
    3. Add water to make up to 1 liter.
• 10× SSC Buffer Preparation:
  • Dilute 500 ml of 20× SSC buffer with water to make 1 liter.

6. Transcription Buffer

Transcription Buffer is used during in vitro transcription reactions.

• 10× Transcription Buffer Preparation:
  • Components (for 10 ml):
    • Tris-HCl, pH 8.0: 400 mM (4 ml of 1 M stock)
    • DTT: 50 mM (0.5 ml of 1 M stock)
    • Triton X-100: 1% (v/v) (1 ml of 10% stock)
    • Spermidine, pH 7.0: 20 mM (0.4 ml of 500 mM stock)
    • MgCl2: 200 mM (2 ml of 1 M stock)
  • Instructions:
    1. Add water to make up to 10 ml.

7. Sodium Phosphate Buffer

Sodium Phosphate Buffer is used to maintain a consistent pH during hybridization.

• Preparation:
  • Components:
    • NaH2PO4: 0.2 M (473.5 ml)
    • Na2HPO4: 0.2 M (26.5 ml)
  • Instructions:
    1. Add water to make up to 1 liter.

8. Denhardt’s Solution

Denhardt’s Solution is used as a blocking agent during hybridization to prevent non-specific binding.

• 100× Denhardt’s Solution Preparation:
  • Components (for 500 ml):
    • Ficoll 400: 0.02 g/ml (10 g)
    • Polyvinylpyrrolidone: 0.02 g/ml (10 g)
    • Bovine Serum Albumin: 0.02 g/ml (10 g)
  • Instructions:
    1. Add water to make up to 500 ml.

9. Hybridization Buffer

Hybridization Buffer facilitates the binding of the probe to the target RNA on the membrane.

• Preparation:
  • Components (for 250 ml):
    • Formamide: 50% (125 ml)
    • SSC: 3× (37.5 ml of 20× stock)
    • Denhardt’s Solution: 10× (25 ml of 100× stock)
    • Sodium Phosphate Buffer, pH 8.0: 10 mM (25 ml of 100 mM stock)
    • EDTA: 2 mM (1 ml of 500 mM stock)
    • SDS: 0.1% (2.5 ml of 10% stock)
    • Salmon Sperm DNA: 200 μg/ml (10 ml of 5 mg/ml stock)
    • Sodium Heparin: 400 U/ml (20 ml of 5000 U/ml stock)
  • Instructions:
    1. Add water to make up to 500 ml.

10. Wash Buffers

Wash Buffers are used to remove non-specifically bound probes during the washing steps.

• Low Stringency Wash Buffer Preparation:
  • Components:
    • SSC: 2× (100 ml of 20× stock)
    • SDS: 0.1% (10 ml of 10% stock)
  • Instructions:
    1. Add water to make up to 1 liter.
• High Stringency Wash Buffer Preparation:
  • Components:
    • SSC: 0.1× (50 ml of 20× stock)
    • SDS: 0.1% (10 ml of 10% stock)
  • Instructions:
    1. Add water to make up to 1 liter.

Northern Blotting Protocol

1. Separation of RNA on a Denaturing Gel

• Preparation of Gel:
  • Add formaldehyde to the agarose solution to prepare the RNA gel solution.
  • Assemble the gel cast and pour the prepared denaturing gel into it.
  • Insert a comb with appropriate teeth to form wells as the gel sets.
• Equilibration and Loading:
  • Once set, remove the comb and equilibrate the gel with running buffer for 30 minutes.
  • Mix 15 µg of RNA sample with an equal volume of RNA loading buffer and add 3 µg of RNA markers in the same buffer.
  • Incubate samples at 65°C for about 12-15 minutes on a heating block.
  • Load the samples and RNA markers into the equilibrated gel wells.
• Electrophoresis:
  • Run the gel at 125V for approximately 3 hours.

2. Transfer of RNA from Gel to Nylon Membrane

• Preparation of Materials:
  • Cut a nylon membrane larger than the denaturing gel.
  • Prepare a filter paper of the same size as the nylon membrane.
• Setting Up the Transfer:
  • After electrophoresis, remove the gel and rinse it with water.
  • Place an oblong sponge slightly larger than the gel in a glass dish and fill with SSC buffer to half-submerge the sponge.
  • Wet a few pieces of Whatman 3mm paper with SSC buffer and place them on top of the sponge.
• Transfer Process:
  • Place the gel on the filter paper and remove air bubbles by rolling a glass pipette over the surface.
  • Wet the nylon membrane with distilled water for 5 minutes, then place it on the gel, avoiding air bubbles.
  • Flood the surface with SSC and place additional filter papers on top.
  • Place a glass plate on the structure to hold it in place and leave it overnight for effective transfer.

3. Immobilization

• Post-Transfer Handling:
  • After transfer, remove the gel and rinse it with SSC, then allow it to dry.
  • Place the membrane between two pieces of filter paper and bake it in a vacuum oven at 80°C for 2 hours.
  • Alternatively, wrap the membrane in UV transparent plastic wrap and irradiate it on a UV transilluminator.

4. Hybridization

• Labeling Probes:
  • Label DNA or RNA probes to a specific activity of >108 dpm/µg and remove unincorporated nucleotides.
• Hybridization Setup:
  • Wet the membrane carrying immobilized RNA with SSC.
  • Place the membrane in a hybridization tube with the RNA side up and add 1 ml of formaldehyde solution.
  • Incubate the tube in a hybridization oven at 42°C for 3 hours.
• Probe Application:
  • If the probe is double-stranded, denature it by heating at 100°C for 10 minutes.
  • Pipette the desired volume of the probe into the hybridization tube and further incubate at 42°C.
• Washing and Detection:
  • Pour off the solution and wash the membrane with a wash solution.
  • Observe the membrane under autoradiography.

Result Interpretation of Northern Blot

1. Visualization of RNA Bands

• Detection:
  • RNA bands are observed as distinct bands on the radiograph.
  • The presence of these bands indicates the successful transfer and hybridization of RNA fragments.
• Band Intensity:
  • The intensity of the bands reflects the amount of RNA present.
  • Stronger bands signify higher concentrations of specific RNA sequences.

2. Comparison with Markers

• Distance Measurement:
  • The distance of RNA bands from the RNA markers is measured.
  • This measurement helps determine the size of RNA fragments.
• Size Determination:
  • By comparing the distance of the bands to the known markers, the length of RNA fragments can be accurately determined.
  • This size determination is crucial for identifying specific RNA transcripts.

3. Semi-Quantification

• Relative Quantification:
  • The intensity of the bands provides a semi-quantitative measure of RNA levels.
  • Comparison of band intensities across different samples can indicate relative RNA abundance.
• Normalization:
  • Normalization against a housekeeping gene can be used to correct for loading variations.
  • This ensures accurate semi-quantitative analysis.

4. Specificity of Hybridization

• Probe Binding:
  • The specificity of the probe hybridization confirms the identity of the RNA fragments.
  • Only RNA sequences complementary to the probe will be detected, ensuring accurate identification.
• Validation:
  • Positive controls and known standards are used to validate the results.
  • This helps in confirming the reliability and accuracy of the interpretation.

5. Functional Insights

• Gene Expression Analysis:
  • The pattern and intensity of bands provide insights into gene expression levels.
  • Differences in expression patterns can indicate various biological conditions or responses.
• Developmental and Tissue-Specific Expression:
  • Analysis can reveal how gene expression varies across different developmental stages or tissues.
  • This information is critical for understanding gene regulation and function.

• Lane 1: RNA sample on 1.2 % denaturing agarose gel
• Lane 2: After Northern hybridization a blue band develops on the nylon membrane 

Precautions

• Prior to start the experiment, the electrophoresis tank should be cleaned with detergent solution (e.g., 0.5% SDS), thoroughly rinsed with RNase-free water, and then rinsed with ethanol and allowed to dry.
• Tips, pipettes, electrophoresis unit etc to be used for the experiment must be UV treated for 15-20 minutes.
• Use sterile, disposable plasticwares and micropipettes reserved for RNA work to prevent cross-contamination with RNases from shared equipments.
• Use RNase-free water for diluting the solutions.

Northern blotting vs Southern blotting

	Northern Blotting	Southern Blotting
Target Molecules	RNA molecules	DNA molecules
Purpose	Detection and analysis of RNA molecules	Detection and analysis of DNA molecules
Technique	Gel electrophoresis, membrane transfer, hybridization	Gel electrophoresis, membrane transfer, hybridization
Target Sequence	Complementary DNA (cDNA) or RNA probes	DNA probes
Applications	Gene expression analysis, RNA processing studies, non-coding RNA analysis	DNA analysis, gene mapping, detection of specific DNA sequences
Detection Method	Radioactive or non-radioactive labeling of probes	Radioactive or non-radioactive labeling of probes
Sensitivity	Generally lower sensitivity compared to newer RNA analysis methods such as qRT-PCR or RNA-seq	Can detect low-abundance DNA sequences, high sensitivity possible with appropriate probes and conditions
Sample Requirements	Requires a relatively large amount of RNA sample	Requires a relatively large amount of DNA sample
Limitations	Time-consuming, lower sensitivity, limited dynamic range for quantification	Time-consuming, potential for DNA degradation, limited dynamic range for quantification
Key Applications	Gene expression analysis, mRNA splicing analysis, non-coding RNA analysis	DNA mapping, detection of specific DNA sequences, analysis of DNA mutations or polymorphisms

Application of northern blotting

• Gene Expression Analysis: Northern blotting is commonly used to study gene expression patterns in different tissues, developmental stages, or disease conditions. By analyzing the abundance of specific mRNA molecules, researchers can determine the level of gene expression and compare it between different samples. This application has contributed to our understanding of gene regulation, cellular processes, and the identification of differentially expressed genes.
• mRNA Splicing Analysis: Northern blotting can provide insights into mRNA splicing patterns. By probing for specific splice variants of a gene, researchers can determine the presence and abundance of alternative mRNA isoforms. This helps in understanding the regulation of alternative splicing events and their functional implications.
• RNA Processing and Stability: Northern blotting allows the investigation of RNA processing events, such as RNA cleavage, polyadenylation, and stability. By examining the size and abundance of specific RNA fragments, researchers can assess the processing efficiency and stability of RNA molecules. This information is crucial for understanding RNA metabolism and post-transcriptional regulation.
• Non-coding RNA Analysis: Northern blotting has been instrumental in the identification and characterization of non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). These non-coding RNAs play important roles in gene regulation, development, and disease. By designing specific probes, researchers can detect and quantify these non-coding RNAs, providing insights into their expression patterns and potential functions.
• Validation of Transcriptomic Data: Northern blotting can serve as a valuable technique for validating transcriptomic data obtained from high-throughput methods, such as RNA sequencing (RNA-seq). It allows the confirmation of gene expression changes observed in large-scale datasets by directly assessing the expression of specific mRNA molecules.
• Diagnostic Applications: Northern blotting has been used in clinical settings for the diagnosis of certain diseases. By detecting the presence and abundance of disease-related RNA molecules or RNA biomarkers, researchers can assess disease status or monitor treatment responses.
• Comparative Analysis: Northern blotting can be utilized to compare RNA expression levels between different samples, such as healthy and diseased tissues, control and experimental conditions, or different individuals. This comparative analysis helps identify molecular differences associated with specific conditions or treatments.

Northern Blotting Advantage

• Detection of Specific RNA Molecules: Northern blotting allows the specific detection and analysis of RNA molecules of interest. By using complementary probes, researchers can target and detect specific RNA sequences, providing insights into gene expression, splicing patterns, and non-coding RNA expression.
• Size Determination: Northern blotting provides information about the size of RNA molecules. By comparing the migration of RNA samples with size markers on the gel, researchers can determine the approximate size of the target RNA molecules, which can be important for understanding RNA processing and transcript variants.
• Validation of Gene Expression Data: Northern blotting can serve as an independent method to validate gene expression data obtained from other techniques, such as RNA-seq. It allows for the confirmation of gene expression changes observed in large-scale datasets by directly assessing the expression of specific mRNA molecules.
• Cost-effective: Compared to some high-throughput techniques like RNA-seq, Northern blotting is a relatively cost-effective method. It does not require expensive equipment or reagents, making it accessible for laboratories with limited resources.
• Customizability: Northern blotting allows researchers to design and generate specific probes for their target RNA molecules. This flexibility enables customization and adaptability to specific research questions or RNA targets of interest.

Northern Blotting Disadvantage

• Time-consuming and Labor-intensive: Northern blotting is a time-consuming technique that requires multiple steps, including RNA extraction, gel electrophoresis, transfer, hybridization, and detection. It involves several manual manipulations and can take several days to complete, making it labor-intensive.
• Low Sensitivity: Compared to newer techniques like quantitative real-time PCR (qRT-PCR) or RNA-seq, Northern blotting may have lower sensitivity. The detection limit of Northern blotting depends on the abundance of the target RNA and the efficiency of probe hybridization. It may not be suitable for detecting low-abundance or rare RNA molecules.
• Limited Dynamic Range: Northern blotting may have a limited dynamic range for quantifying RNA expression. The intensity of the hybridization signal on the blot does not always correspond linearly to the abundance of RNA in the sample. It may be challenging to accurately quantify RNA expression levels using Northern blotting.
• Sample Degradation: RNA is prone to degradation by RNases, making it necessary to handle RNA samples with care. Contamination or improper handling can lead to RNA degradation, affecting the quality and integrity of the RNA samples and potentially compromising the results of Northern blotting.
• Relatively Large Sample Requirements: Northern blotting typically requires a relatively large amount of RNA sample, making it challenging when working with limited or precious sample materials. Extraction of sufficient RNA can be difficult, particularly for small or rare cell populations.

Northern Blotting Cheatsheet/Infographic

FAQ

References

• He SL, Green R. Northern blotting. Methods Enzymol. 2013;530:75-87. doi: 10.1016/B978-0-12-420037-1.00003-8. PMID: 24034315; PMCID: PMC4287216.
• https://en.wikipedia.org/wiki/Northern_blot
• https://himedialabs.com/TD/HTBM028.pdf
• https://www.thesciencenotes.com/northern-blotting-principle-procedure-and-applications/
• https://www.mybiosource.com/learn/testing-procedures/northern-blotting/