Efficient and rapid isolation of mRNA is important in thefield of genomics as well as in the clinical and pharmaceutical arena. We have developed UV-initiated methacrylate-based porous polymer monoliths (PPM) for microfluidic trapping and concentration of eukaryotic mRNA. PPM are cast-to-shape and are tunable for functionalization using a variety of amine-terminated molecules. Efficient isolation of eukaryotic mRNA from total RNA was first mathematically modeled and then achieved using PPM in capillaries. Purification protocols using oligo dT's, locked nucleic acid substituted dT's, and tetramethylammonium chloride salts were characterized. mRNA yield and purity were compared with mRNA isolated by commercial kits with statistically equivalent yields and purities (determined by qPCR ratio of 18s rRNA and Gusb mRNA markers). Even after extracting 16 (mu)g of mRNA from 315 (mu)g of total RNA, the 0.4-(mu)L volume monolith showed no signs of saturation. Elution volumes were below 20 (mu)L with concentrations up to 1 (mu)g/(mu)L. In addition, the polymeric material exhibited exceptional stability in a range of conditions (pH, temperature, dryness) and was stable for a period of months. All of these characteristics make porous polymer monoliths good candidates for potential microfluidic sample preconcentrators and purifiers.
展开▼
