Question

Which of the following methods could be used to enrich the mRNA species of the total RNA population extracted from a plant tissue?

• A. Separation of total RNA on a polyacrylamide gel
• B. Affinity chromatography using oligo(dT)-cellulose
• C. Precipitation of total RNA using organic solvents
• D. Employing salting-in and salting-out principle

Hint:

Think of Oligo(dT) as a "Magnetic Hook" for mRNA.
It works because mRNA is the only RNA type with a "Poly-A" tail.
This step is essential before constructing a cDNA library or performing RNA-seq.

Answer 1

The Correct Option is B

Step 1: Understanding the Question:
Total RNA extracted from a cell consists mostly of ribosomal RNA (rRNA, ~80-85%) and transfer RNA (tRNA, ~10-15%), with messenger RNA (mRNA) making up only about 1-5%. The question asks how to specifically isolate or "enrich" the mRNA fraction from this mixture.

Step 2: Detailed Explanation:

• Structural Basis for Isolation: Eukaryotic mRNA (including that from plants) has a unique structural feature: a poly-adenylated (poly-A) tail at its 3' end. This tail consists of a long string of adenine nucleotides.

• Affinity Principle: Oligo(dT)-cellulose contains short chains of deoxythymidine (T) fixed to a solid cellulose support. Because Adenine pairs with Thymine through hydrogen bonds, the poly-A tails of mRNA molecules will hybridize (bind) to the oligo(dT) chains.

• The Procedure: Total RNA is passed through a column containing oligo(dT)-cellulose under high-salt conditions. The mRNA sticks to the column, while rRNA and tRNA (which lack poly-A tails) pass through and are washed away.

• Elution: The bound mRNA is then recovered (eluted) by passing a low-salt buffer or water through the column, which breaks the A-T hydrogen bonds.

• Analysis of other options:
- Electrophoresis (A) separates RNA by size but is not practical for large-scale enrichment of diverse mRNA species.
- Precipitation (C) recovers all RNA types indiscriminately.
- Salting-out (D) is a technique used primarily for protein purification, not RNA fractionation.

Step 3: Final Answer:
Affinity chromatography utilizing the complementary base pairing between mRNA poly-A tails and oligo(dT) ligands is the standard method for mRNA enrichment.