The purification of poly(a)-containing RNA by affinity chromatography

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Most eukaryotic messenger RNA (mRNA) molecules possess a poly(adenylic) acid tail at their 3' end.
• This poly(A) tail can be up to 250 nucleotides long.
• The presence of the poly(A) tail is a conserved feature across eukaryotic mRNA.

Purpose of the Study:

• To highlight the utility of the poly(A) tail for mRNA extraction.
• To describe a simple and convenient affinity chromatography method for mRNA isolation.
• To explain the principle behind poly(A)-based mRNA purification.

Main Methods:

• Utilizing the hybridization property of poly(A) sequences with complementary oligo(dT)-cellulose or poly(U)-Sepharose under high salt conditions (0.3-0.5M NaCl or KCl).
• Employing commercially available affinity matrices containing polymers of 10-20 nucleotides bound to a carbohydrate support.
• Exploiting the specific binding of poly(A) tracts as short as 20 residues.

Main Results:

• The poly(A) tail of mRNA specifically binds to oligo(dT)-cellulose or poly(U)-Sepharose.
• Ribosomal RNA and transfer RNA, lacking poly(A) sequences, do not bind to these matrices.
• This method allows for the selective isolation of mRNA.

Conclusions:

• The poly(A) tail of eukaryotic mRNA provides a robust basis for affinity chromatography-based purification.
• This technique offers a simple and effective method for isolating mRNA, distinguishing it from other RNA species.
• The described method is valuable for molecular biology applications requiring pure mRNA samples.