Northern Blot Method - Animated Video

Northern Blot Technique in Molecular Biology

The northern blot technique is a classic method used in molecular biology to study gene expression by detecting isolated mRNA in a sample. This section provides an overview of the technique and its steps.

Preparation of RNA Samples

• Formaldehyde is added to the samples for denaturation of the RNA molecules, which helps maintain them in a denatured state.

• Self-complementary sequences in the RNA strand lead to intrachain base pairing and folding into complex structural forms known as secondary structures.

• Gel electrophoresis is used to separate the RNA samples based on their charge and size.

Gel Electrophoresis

• A loading buffer with tracking dye is added to monitor the progress of separation.

• Agarose gel electrophoresis is commonly used for separating mixtures of RNA molecules.

• A molecular weight size marker (RNA ladder) helps determine the size of RNA molecules.

Staining and Transfer

• After electrophoresis, the RNA molecules in the gel can be stained using intercalating dyes like ethidium bromide.

• Northern blotting is performed to transfer the RNA molecules from the gel onto a membrane.

• Capillary action and ion exchange interactions facilitate this transfer process.

Hybridization with Probes

• The transferred RNA on the membrane is attached permanently using ultraviolet radiation.

• Radio-labeled probes are then hybridized with the attached RNA on the membrane for specific detection.

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New Section DNA Probe Hybridization Process

This section explains the process of hybridizing radioactively labeled DNA probes to their complementary sequences in RNA molecules.

Hybridization and Removal of Hybridization Solution

• Radioactively labeled DNA probes are hybridized to their target sequences in RNA molecules.

• After hybridization, the hybridization solution is removed.

Washing Process

• A wash buffer is added to remove unbound and weakly binding probes.

• The membrane is incubated at 52 degrees Celsius for 30 minutes.

• The washing process is repeated three times.

Autoradiography Method

• An autoradiography method is carried out to identify the location of radioactively labeled RNA in the membrane.

• The Southern blot filter is placed inside a light-proof cassette box.

• An X-ray film is laid over the top of the cassette.

• The cassette is closed and left for several hours to several days.

Result Interpretation

• The radioisotope-labeled RNA exposes the film, which, when developed, shows a pattern of black bands.

• These black bands indicate the positions of labeled RNA in the blot membrane.