Introduction

Northern blotting is a molecular biology technique used to detect, identify, and study RNA molecules (mainly mRNA) within a sample.

• It was developed by James Alwine, David Kemp, and George Stark in 1977.

• The name “Northern blot” was coined as an analogy to the Southern blot (a method for DNA detection developed by Edwin Southern).

• Since it focuses on RNA, it plays a crucial role in gene expression studies because mRNA reflects which genes are actively being transcribed in a cell or tissue at a given time.

👉 In short:
Northern blotting is a hybridization-based method that separates RNA by size and detects specific RNA sequences using complementary nucleic acid probes.

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Principle

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The principle of Northern blotting relies on nucleic acid hybridization:

1. RNA Separation:

   • RNA molecules are separated by size using agarose gel electrophoresis (with formaldehyde or glyoxal to denature RNA).

2. Transfer to Membrane:

   • The separated RNA fragments are transferred to a nylon or nitrocellulose membrane.

3. Fixation:

   • RNA is immobilized on the membrane by UV cross-linking or baking.

4. Hybridization with a Labeled Probe:

   • A single-stranded DNA or RNA probe, complementary to the target RNA sequence, is labeled (radioactive, fluorescent, or chemiluminescent).

   • This probe hybridizes specifically to its complementary RNA sequence on the membrane.

5. Detection:

   • The hybridized probe is detected by autoradiography, fluorescence, or chemiluminescence.

   • The signal corresponds to the presence, size, and abundance of the RNA of interest.

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Steps of Northern Blotting

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Step 1: Sample Preparation

• Extract total RNA or mRNA from cells/tissues using methods such as TRIzol reagent, phenol–chloroform extraction, or column-based kits.

• RNA integrity is checked using agarose gel electrophoresis or a bioanalyzer.

• RNA is very sensitive to RNases, so all steps must be performed with RNase-free reagents and equipment.

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Step 2: Gel Electrophoresis

• RNA samples are denatured to prevent secondary structure formation.

• They are separated on agarose gel containing formaldehyde or glyoxal (denaturing agents).

• Separation is based on size (nucleotide length).

• Ethidium bromide or SYBR Green may be used to visualize RNA migration.

📌 Example: A 2 kb mRNA will migrate slower than a 0.8 kb mRNA.

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Step 3: Transfer to Membrane

• After electrophoresis, RNA is transferred from the gel to a nylon or nitrocellulose membrane.

• Methods:

  • Capillary transfer (classic method): Buffer moves upward by capillary action, carrying RNA from gel to membrane.

  • Vacuum or electroblotting: Faster, more efficient alternatives.

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Step 4: Fixation

• RNA molecules are covalently bound to the membrane:

  • UV Crosslinking (most common): RNA is exposed to UV light.

  • Baking: Membrane heated at 80 °C.

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Step 5: Hybridization with Probe

• A labeled probe (complementary to target RNA) is prepared.

• Types of probes:

  • Radioactive probes (e.g., ³²P-labeled DNA or RNA).

  • Non-radioactive probes (fluorescent, digoxigenin (DIG)-labeled, biotin-labeled).

• Membrane is incubated with probe under conditions that allow specific base pairing (hybridization).

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Step 6: Washing

• Excess unbound probe is removed by washing with buffer under stringent conditions.

• Ensures only specific hybridization signals remain.

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Step 7: Detection

• Depending on the probe label:

  • Autoradiography: X-ray film detects radioactive probe.

  • Fluorescence Imaging: For fluorescent probes.

  • Chemiluminescence: Probe coupled with enzyme that emits light upon substrate addition.

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Step 8: Analysis

• The detected signal appears as bands on film or an imaging system.

• Band position: Indicates RNA size (compared to RNA ladder).

• Band intensity: Reflects relative abundance of mRNA.

📌 Example: If a cancer cell expresses more c-myc mRNA, a stronger band will appear in tumor samples compared to normal samples.

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Results Interpretation 

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• Presence/absence of bands: Confirms whether the target RNA is expressed.

• Size of band: Indicates transcript length (e.g., splicing variants, processing events).

• Band intensity: Provides relative measure of transcript abundance.

• Multiple bands: May indicate different isoforms or alternative splicing products.

• Controls used:

  • Loading controls (e.g., 18S rRNA, GAPDH mRNA): To ensure equal RNA loading across lanes.

  • Negative controls: To check probe specificity.

  • Positive controls: To confirm probe functionality.

 

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Applications 

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1 Gene Expression Studies

• Detects specific mRNA levels in different cell types, tissues, or developmental stages.

• Example: Comparing β-globin mRNA in fetal vs. adult tissues.

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2 Alternative Splicing Analysis

• Identifies splice variants by revealing transcripts of different sizes.

• Example: Tropomyosin gene produces multiple isoforms; Northern blot distinguishes them.

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3 RNA Processing Studies

• Studies pre-mRNA splicing, polyadenylation, and degradation.

• Example: Detecting unspliced vs. spliced transcripts in mutant cells.

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4 Verification of Gene Expression 

• Confirms whether a cloned gene is transcribed in transfected cells.

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5 Disease Diagnosis and Research

• Used to detect viral RNAs in infections.

• Example: Detecting hepatitis C virus RNA in patient samples.

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6 Drug Development

• Helps study changes in gene expression upon drug treatment.

• Example: Testing whether a chemotherapy drug downregulates oncogene mRNAs.

 

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Advantages 

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• Provides information on RNA size and abundance.

• Highly specific (probe-based detection).

• Can detect alternative splicing and isoforms.

• Useful for validating results of other techniques (RT-PCR, microarrays).

• Stable record (blots can be stored and re-probed).

 

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Limitations 

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• Labor-intensive and time-consuming.

• Requires large amounts of RNA compared to RT-PCR.

• Lower sensitivity compared to qRT-PCR or RNA-seq.

• Use of radioactivity poses safety concerns.

• Requires high-quality, intact RNA (easily degraded by RNases).

 

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Comparison with Other Techniques

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