4-3.2.1. Isolation of mRNA

It involves the isolation of total mRNA from a cell type or tissue of interest. The amount of desired mRNA can be increased by following ways-

•  Chromatographic purification of mRNA using oligo-dT column, which retains mRNA molecules, resulting in their enrichment.

•  Spinning down mRNA by density gradient centrifugation.

•  mRNA preparation from specialized cell types, e.g. developing seeds, chicken oviduct, erythrocytes, β cells of pancreas etc.

The 3′ ends of eukaryotic mRNA consist of a string of 50 – 250 adenylate residues (poly A Tail) which makes the separation easy from the much more prevalent rRNAs and tRNAs in a cell extract using a column containing oligo-dTs tagged onto its matrix.

When a cell extract is passed through an oligo-dT column, the mRNAs bind to the column due to the complementary base-pairing between poly (A) tail and oligo-dT. Other RNAs (ribosomal RNAs and transfer RNAs) flow through as unbound fraction. The bound mRNAs can then be eluted using a low-salt buffer.