Procedures such as cDNA synthesis, expression profiling and others require separation of mRNA from the vastly more abundant rRNA and tRNA. The GenElute mRNA kits provide convenient procedures for isolating polyadenylated mRNA from previously prepared total RNA or directly from mammalian cells and tissues. For direct mRNA preparation, cells or tissues are disrupted with SDS/proteinase K digestion to release RNA and eliminate RNases. Both kit types use oligo dT30 covalently linked to 1 μm polystyrene beads to capture polyadenylated mRNA by hybridization. The polystyrene beads remain suspended during hybridization, eliminating the need for mixing or rocking, as is common for cellulose or magnetic particles. Polystyrene was also chosen because oligo(dT) polystyrene beads yield cleaner mRNA with fewer stringent washing steps than does the more commonly used oligo(dT) cellulose (2 or 3 wash steps versus 10 or more). With the GenElute kits, mRNA-bead complexes are washed on a microcentrifuge spin filter, and eluted into 10 mM Tris-HCL, pH 7.5. mRNA prepared with either kit is suitable for a variety of downstream applications such as Northern blotting, expression array or chip hybridizations and cDNA synthesis and library construction.
The purified mRNA is ready for Northern analysis, reverse transcription and PCR, labeling for arrays, and other common applications.