Protocol for mRNA Northern Blotting

Northern blotting is a fundamental molecular biology technique used to study gene expression by detecting specific RNA molecules within a complex mixture. Unlike Southern blotting (which detects DNA) or Western blotting (which detects proteins), Northern blotting provides information about RNA size, abundance, and integrity.

Overview of the Procedure

The Northern blot process involves the separation of RNA samples by size using gel electrophoresis, followed by the transfer of the separated RNA onto a membrane (typically nylon or nitrocellulose) and subsequent detection using a labeled hybridization probe.

Step 1: RNA Preparation and Gel Electrophoresis

Because RNA is highly susceptible to degradation by RNases, it is critical to use RNase-free water, reagents, and plasticware throughout the procedure. DEPC-treated water is standard.

RNA Extraction: Isolate total RNA or mRNA from cells or tissue samples using established methods (e.g., Trizol or column-based kits).
Denaturing Gel Preparation: Prepare an agarose gel containing formaldehyde. Formaldehyde acts as a denaturant to disrupt secondary structures in the RNA, ensuring that separation is based strictly on molecule length.
Sample Loading: Mix RNA samples with a denaturing loading buffer containing formamide. Heat the samples to approximately 65C for 5-10 minutes before loading into the gel.
Electrophoresis: Run the gel at a constant voltage in a running buffer containing formaldehyde.

Step 2: RNA Transfer (Blotting)

Once electrophoresis is complete, the RNA must be transferred from the gel to a solid support membrane.

Capillary Transfer: The most common method involves placing the gel in contact with a nylon membrane, topped with a stack of paper towels. The transfer buffer moves by capillary action from the bottom, carrying the RNA out of the gel and onto the membrane.
Fixation: Once transferred, the RNA must be immobilized on the membrane. This is typically achieved through UV cross-linking or baking the membrane at 80C for two hours.

Step 3: Pre-hybridization and Hybridization

Hybridization allows the probe to bind to its complementary RNA sequence on the membrane.

Pre-hybridization: Incubate the membrane in a buffer (e.g., Church-Gilbert buffer or ExpressHyb) to block non-specific binding sites on the membrane surface.
Probe Labeling: Prepare a labeled probe, typically using radioactive isotopes (e.g., 32P) or non-radioactive markers like digoxigenin (DIG) or biotin.
Hybridization: Add the labeled probe to the hybridization buffer and incubate the membrane at a temperature determined by the probe's melting temperature (Tm), usually between 42C and 68C.

Step 4: Washing and Detection

After hybridization, it is necessary to remove unbound or weakly bound probes to minimize background noise.

Washing: Perform several high-stringency washes using buffers with varying salt concentrations (SSC) and SDS. Higher temperatures and lower salt concentrations increase stringency.
Detection: If using radioisotopes, expose the membrane to X-ray film or a phosphorimager screen. If using non-radioactive probes, use enzymatic detection methods, such as anti-DIG antibodies conjugated to alkaline phosphatase, followed by chemiluminescence development.

Critical Success Factor: The quality of RNA is the most important factor in a successful Northern blot. Always ensure the samples are intact by checking for clear 28S and 18S ribosomal bands on the gel before proceeding to transfer.

Troubleshooting Common Issues

High Background: Often caused by insufficient blocking or non-specific probe hybridization. Increase the wash stringency.
Faint or No Signal: May indicate probe degradation, poor RNA quality, or inadequate hybridization time.
Smearing: Indicates RNA degradation. Ensure all materials are RNase-free and consider using fresh RNA samples.

Reference Files For MRNA Northern Blot Protocol

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