Fractionation of Small RNA by HPLC

For all functional analyses, it is necessary to purify mRNA from the other types of RNA. mRNA constitutes only a small fraction (a few percent) of the total RNA. For separation of mRNA from the rest of RNA, advantage is taken of the fact that most mRNA species have a long poly-A+ tail at their 3 end (2-3). Oligo(dT) or poly-U affinity matrix is used to bind poly A+-containing mRNA that can then be eluted from the column. Several such methods exist with variations in the type of oligo(dT) used or the matrix to which it is attached. 

Isolation of mRNA Using Oligo(dT)-Cellulose Matrix 

Isolation of mRNA by Biotin-Streptavidin Affinity Method 

Bind poly A+ RNA from total RNA and capture poly A+ RNA-oligo(dT) hybrid on streptavidin beads. 

Centrifuge the beads. Wash and elute the poly A+ RNA with water 

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