RNA based therapeutics represent a powerful new tool for gene therapy. In RNA production, impurities such as nucleotide triphosphates, abortive transcripts, and DNA template can affect down-stream applications by triggering immune responses. Gel-based methods of purification, such as preparative denaturing polyacrylamide gel electrophoresis (PAGE) and agarose gels, are limited by poor resolution of RNA size and potential chemical modifications from reagents such as formaldehyde. To date, high performance liquid chromatography (HPLC) remains a staple method for purification of messenger RNA (mRNA) and RNA oligonucleotides. Presented here are two HPLC methods, a size exclusion-based method for the purification of long mRNA, and a reverse-phase method for the purification of oligonucleotides.
Figure 1. Molecular weight determination for mRNA up to 5,000 bases