Plant Lab Protocols / Methodology for Nucleic Acids
Isolation of messenger RNA by affinity chromatography
Messenger RNA (mRNA) accounts for only about 2% of the total RNA in plants, the rest being occupied by tRNA and rRNA. For a long time it was believed that mRNA cannot be separated from the bulk RNA. However, there was a breakthrough after the discovery that only messenger RNA carries a stretch of polyadenylate at its 3' end. The polyadenylate 'tail' may vary in length ranging 3-200 adenylate residues.
Principle
Under high ionic conditions, the poly(A) tail of mRNA forms a duplex with complementary base sequences such as poly(U) or oligo(dT) while tRNA and rRNA do not form any duplex since they lack poly(A) tail. When the ionic condition is lowered the hydrogen bonding between poly (A) and poly(U) or oligo(dT) is broken, thus releasing the Poly (A) containing molecules. This is the basis for the development of affinity chromatography to isolate the messenger RNA from the bulk RNA. Poly(U) or oligo(dT) immobilized (covalently attached) to sepharose or cellulose is used as the stationary phase.
Materials
» Oligo(dT) Cellulose or Poly(U) Sepharose
» Binding Buffer (pH 7.5)
0.01M Tris 0.12g
0.5M NaCl 2.92g
0.001M EDTA Na2 0.04g adjust pH with HCl
10% SDS 0.5mL
Water 100mL
» Add SDS solution to the buffer before use.
» Elution Buffer (pH 7.5)
0.01M Tris 0.12g
0.001M EDTA Na2 0.04g adjust pH with HCl
10% SDS 0.5mL
Water 100mL
» Spectrophotometer
» Chromatography Column (Pasteur Pipette)
All glassware and solutions should be sterile.
Procedure
1.
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Suspend about 500mg of affinity material oligo-(dT) cellulose or Poly-(U) Sepharose) in 20mL of sterile binding buffer.
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2.
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Pack the column with the affinity material as usual and equilibrate it by passing through 25-30mL of binding buffer.
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3.
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Dissolve the isolated total RNA in the binding buffer at a concentration of approximately one mg per mL.
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4.
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Apply the RNA solution to the column. Recycle the initial effluent of the column.
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5.
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Wash the column extensively with the binding buffer until the absorbance at 260nm reaches zero. The poly (A) containing mRNA is retained in the column while tRNA and rRNA pass through. Collect the high absorbance initial fractions and precipitate with ethanol for rRNA and tRNA, if desired.
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6.
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Elute the column with low salt sterile elution buffer. Collect and pool high A260 fractions. These fractions contain mainly mRNA with slight contamination of rRNA.
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7.
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Wash the column with elution buffer and then pre-equilibrate with the binding buffer.
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8.
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Combine and heat the eluted fractions (Step 6) at 55°C for 5min in order to disrupt the aggregation, cool sudden and add solid NaCl to a concentration of 0.5M.
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9.
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Apply this solution to pre-equilibrated column (Step 7) with the binding buffer.
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10.
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Repeat steps 5 and 6. The eluted high A^ fractions are pooled, and made 0.5M NaCl by adding solid substance. The RNA is precipitated, after adding two volumes of ice-cold ethanol, at -20°C for at least 20h.
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11.
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The poly(A) rich RNA (mRNA) is collected, dissolved and estimated spectrophotometrically as described under ‘Isolation of RNA' (Step 12).
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Notes
1.
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All the glasswares should be sterile (baked at 120°C overnight). The solutions should be sterilized by autoclaving at 20lb/cm2 for 20 min.
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2.
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The dissolution of RNA in the binding buffer is slow due to high salt concentration. On the other hand, RNA is first dissolved easily in the elution buffer and then made to 0.5M NaCl by adding solid substance.
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3.
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After the elution of bound mRNA, the affinity material is charged by passing through 0.1 N NaOH and stored in the binding buffer containing 0.1% sodium azide at 0-4°C.
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References
1. Aviv, H and Leder, P (1972) Proc NatlAcad Sci USA 691408.
2. Bantle, J A, Maxwell, IH and Hahn, W E (1976) AnalBiochem 72 413-427.
3. Manual on Techniques in Molecular Biology' (Nucleic Acids) (1986) Workshop held at the Department of Biochemistry, Tamil Nadu Agricultural University Coimbatore p 40.
4. Mam'ckam, A, Peumans, W J and Carlier, A R (1979) Arch int Physiol Biochem 87 195. |
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