Q.19 Which one of the following technique is used for the characterisation of RNA?
(1) Western blotting
(2) Southern blotting
(3) Northern blotting
(4) Eastern blotting
Blotting Techniques: The Molecular Biology Workhorses
Blotting methods transfer biomolecules from gels to membranes for detection via probes. Named after targets, they’re essential for gene expression studies. This MCQ zeros in on RNA: Which one of the following technique is used for the characterisation of RNA? (1) Western blotting (2) Southern blotting (3) Northern blotting (4) Eastern blotting.
Correct Answer: (3) Northern blotting
Northern blotting specifically sizes, quantifies, and identifies RNA molecules.
How Northern Blotting Works for RNA Characterization
Developed by James Alwine (1977), it detects specific RNA sequences:
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Isolation & Electrophoresis: Total RNA run on denaturing agarose gel (formaldehyde/formamide prevents secondary structure).
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Transfer (Blotting): RNA capillary-blotted to nitrocellulose/nylon membrane.
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Hybridization: Labeled DNA/RNA probe binds target mRNA via complementarity.
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Detection: Autoradiography, chemiluminescence reveals bands; size via RNA ladder.
Uses: Quantify gene expression (e.g., oncogenes), detect splicing variants. Modern alternatives like RT-qPCR are faster but Northern confirms RNA integrity.
Full Breakdown of All Options: Target by Target
Memorize by mnemonic: Western (Western = Protein), Southern (S = DNA), Northern (N = RNA).
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(1) Western blotting: For proteins. SDS-PAGE separates by size, antibody detection. Not RNA.
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(2) Southern blotting: For DNA. Agarose gel, restriction digest, DNA probe. Detects genes/RFLPs.
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(3) Northern blotting: For RNA. As above—sizes transcripts, checks poly-A tails.
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(4) Eastern blotting: Not standard for RNA (or routine use). Emerging term for proteins (native, no SDS) or glycoproteomics. Rarely for RNA; sometimes confused with “Far Eastern” for lipids.
| Technique | Target Molecule | Gel Type | Probe Type | Key Application |
|---|---|---|---|---|
| Western | Proteins | SDS-PAGE | Antibodies | Protein levels/post-translational mods |
| Southern | DNA | Agarose | DNA | Gene mapping, mutations |
| Northern | RNA ✓ | Denaturing agarose | DNA/RNA | Transcript size/expression |
| Eastern | Proteins/glycans | Native PAGE | Lectins/antibodies | Native proteins, glycosylation |
Why Northern Matters in Modern Genomics
Despite RNA-seq dominance, Northern blotting validates results (e.g., miRNA studies). It’s gold standard for RNA size accuracy. Pro tip: RNA’s instability requires RNase-free protocols.
Structure overview:
Gel → Blot → Hybridize → Detect
RNA → Membrane → Probe → Signal
Resources: Sambrook’s Molecular Cloning or protocols from Thermo Fisher.
