Northern Blotting Technique

 Northern blotting is a laboratory method used to detect specific RNA sequences in a sample. It is named by analogy to Southern blotting, which is used for DNA detection. The technique helps in understanding gene expression by analyzing RNA (or mRNA) patterns.

Steps of Northern Blotting:

1. RNA Extraction: RNA is extracted from cells or tissues using appropriate techniques.

2. Gel Electrophoresis: The extracted RNA is separated based on size using agarose gel electrophoresis under denaturing conditions (often with formaldehyde to prevent secondary structures in RNA).

3. Transfer to Membrane: After electrophoresis, the RNA is transferred from the gel onto a nylon or nitrocellulose membrane through capillary action or electroblotting.

4. Hybridization with Probe: The membrane is incubated with a labeled probe (radioactive, fluorescent, or chemiluminescent). The probe is a short sequence of nucleotides that is complementary to the RNA of interest.

5. Detection: After hybridization, the unbound probe is washed away, and the labeled RNA is detected using an appropriate detection system (e.g., autoradiography for radioactive probes, chemiluminescence, or fluorescence).

Applications:

• Gene Expression Studies: It helps determine when and where specific genes are expressed by analyzing RNA levels.
• RNA Integrity Assessment: Useful in checking the quality and integrity of RNA samples.
• Detection of Alternative Splicing: Identifies different RNA isoforms produced by alternative splicing.

Northern blotting is valued for its specificity, though newer techniques like RT-qPCR and RNA sequencing are becoming more common due to higher sensitivity and throughput.