Northern

Northern blotting is a laboratory technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. With its ability to reveal the size and relative abundance of specific RNA molecules, Northern blotting serves as a crucial step in understanding the molecular underpinnings of various biological processes and diseases.

Overview

Northern blotting involves the separation of RNA samples by gel electrophoresis, followed by their transfer to a membrane (typically nylon or nitrocellulose) and subsequent hybridization with labeled probes that are complementary to the target RNA sequence. This method allows researchers to detect specific RNA molecules within a complex mixture, making it an invaluable tool for studying gene expression patterns, RNA processing, and viral infections.

Procedure

The Northern blotting procedure can be divided into several key steps:

1. RNA Isolation: Total RNA is extracted from the tissue or cells of interest using various chemical or enzymatic methods.
2. Gel Electrophoresis: The isolated RNA is then separated based on size by agarose gel electrophoresis. RNA samples are typically denatured prior to loading to ensure that they run according to their linear length.
3. Transfer to Membrane: The separated RNA molecules are transferred from the gel to a solid support membrane, a process known as blotting. This is usually achieved through capillary action or electric transfer.
4. Probe Hybridization: A labeled DNA or RNA probe, complementary to the target RNA sequence, is then hybridized to the RNA on the membrane. Probes can be labeled with radioactive isotopes, fluorescent dyes, or enzymes for detection.
5. Detection: The location and intensity of the probe signal on the membrane are detected and analyzed, providing information on the size and abundance of the target RNA.

Applications

Northern blotting is used in various research and clinical settings to:

• Study gene expression levels under different conditions or treatments.
• Analyze RNA processing events, such as splicing, editing, and polyadenylation.
• Detect and quantify viral RNA in infection studies.
• Investigate the function of non-coding RNAs, such as microRNAs and long non-coding RNAs.

Advantages and Limitations

While Northern blotting is a powerful tool for RNA analysis, it has its advantages and limitations. It provides direct evidence of RNA size and abundance, and the use of specific probes ensures high specificity. However, the technique is relatively labor-intensive and requires a significant amount of RNA. Moreover, newer techniques such as real-time PCR and RNA-Seq offer higher sensitivity and throughput, leading to a decline in the use of Northern blotting for some applications.

See Also

• Molecular biology
• Gene expression
• RNA
• Gel electrophoresis
• Blotting

References

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