Automated High-Throughput mRNA Selection From Eukaryotic Total RNA

We describe a new technology (patent pending) for high-throughput selection of poly(A)+ RNA from total RNA. A novel binding solution is used to ensure the efficient and specific binding of mRNA to oligo(dT) magnetic beads with high stringency, virtually eliminating the non-specific binding of ribosomal RNA (rRNA) either to oligo(dT) beads or to the poly(A)+ RNA bound to the beads. As quantified by real-time RT-PCR, more than 99% of the rRNA is removed in a single round selection and mRNAs are fully recovered for both highly-expressed (GAPDH) and poorly-expressed (DDPK) genes from a few μg total RNA. The protocol is adaptable to any generic robotic workstation and takes ~30 minutes to process 96 samples.