Lecture on Northern blotting technique

1. Northern Blotting: Principle,
Procedure, and Applications

2. Introduction
• Developed in the 1970s as an adaptation of
Southern blotting
• Used for detection of specific RNA sequences
• Helps study gene expression at the
transcriptional level

3. Why Use Northern Blotting?
• Detects and quantifies specific mRNA
molecules
• Analyzes gene expression patterns
• Monitors transcriptional activity in different
tissues or conditions
• Confirms RNA integrity and size

4. Principle of Northern Blotting
• RNA is extracted and separated by gel
electrophoresis
• Transferred to a membrane (usually nylon)
• Hybridized with a labeled nucleic acid probe
• Probe binds to complementary RNA sequence
• Detected using autoradiography or
chemiluminescence

5. Materials and Reagents
• Total or poly(A)+ RNA
• Formaldehyde-agarose gel
• Blotting membrane (nylon)
• Labeled DNA or RNA probe
• Hybridization and washing buffers
• Detection reagents

6. Steps in Northern Blotting
• 1. RNA Extraction
• 2. Gel Electrophoresis (denaturing conditions)
• 3. Transfer to Membrane (Blotting)
• 4. Fixation of RNA to Membrane
• 5. Hybridization with Labeled Probe
• 6. Washing and Detection

7. Applications
• Gene expression analysis
• Tissue-specific and developmental studies
• Verification of RNA size and transcript variants
• Detection of RNA splicing patterns
• Validation of RNA-seq data

8. Advantages and Limitations
• Advantages:
- Direct visualization of transcript size and
abundance
- Specific and reliable for transcript detection
• Limitations:
- Time-consuming and labor-intensive
- Requires high-quality RNA
- Less sensitive than RT-PCR or RNA-seq
- Limited throughput

9. Modern Alternatives
• RT-PCR / qRT-PCR – Sensitive and quantitative
• RNA-seq – High-throughput transcriptome
profiling
• Microarrays – Gene expression screening
• In situ hybridization – Spatial gene expression
analysis

10. Conclusion
• Northern blotting remains a classic tool for
RNA analysis
• Still used to confirm transcript size and
integrity
• Often complemented or replaced by modern,
high-throughput methods