Purification of mRNA directly from crude plant tissues in 15 minutes using magnetic oligo dT microspheres.

Most strategies for mRNA isolation involve as a first step isolation of total RNA and as a second step selection of poly A + RNA by affinity chromatography on oligo dT cellulose columns. Here we report a novel method which allows isolation of poly A + RNA directly from crude plant tissue without first preparing total RNA or any other purification steps. The method is based upon the addition of magnetic monosized microspheres with covalently attached oligo (dT)^ residues directly to the crude lysate. Using this method poly A+ RNA can be prepared successfully from several tissues from barley (Hordeum vulgare) such as embryo, aleurone, seedlings, roots and leaves. The RNA prepared with this method can be used without further purification for Northern hybridization (Figure 1), in vitro translation (Figure 2) as well as cDNA synthesis (not shown). The method is extremely fast; starting from crude frozen plant tissue it takes less than 15 min to prepare poly A+ RNA. Method: Tissue (0.1-0.2 g) frozen in liquid nitrogen was ground to a fine powder. The frozen powder was transferred to a Duall homogenator containing 1.2 ml lysis/poly A-binding buffer (0.5 M LiCl, 0.1 M Tris pH 8.0, 0.01 M EDTA, 1% LiDS and 5 mM DTT) and homogenized. The lysate was spun 30 sec. (10,000 g) in a microfuge, and the supernatant transferred to a microfuge tube containing 2 mg of Dynabeads oligo dT (Dynal) suspended in 100 /J lysis/poly A-binding buffer. The RNA was annealed to the beads for 4 5 min at room temp or on ice. The beads were collected by placing the microfuge tube on a magnet (MPC-E, Dynal) and removing the supernatant. Beads were washed and resuspended 3 times in 1 ml 0.15 M LiCl, 0.01 M Tris pH 8.0, 1 mM EDTA and 0.1 % SDS. Final elution was in distilled water for 2 min at 55 °C.