Cleaning up in vitro transcribed RNA

In vitro transcribed RNA also often needs to be cleaned up and separated from components such as DNA templates, unincorporated nucleotides or RNA modifying enzymes. This is especially true following in vitro transcription, generation of sgRNA for genome editing, and to remove DNase following DNase I treatments. The purification method selected depends on RNA length and abundance, downstream applications and whether the contaminating species are nucleic acids and/or proteins. For recovery of RNAs greater than 300 nucleotides long, lithium chloride precipitation can be employed to remove the majority of unincorporated nucleotides and enzymes.  To obtain high purity RNA, gel purification is recommended to ensure removal of free nucleotides and truncated transcription products, though recovery may be reduced.  Spin column purification is the desired method to remove unincorporated nucleotides, proteins and salts, though columns have varying size cut-offs and loading capacities. NEB’s Monarch® Nucleic Acid Purification portfolio includes products for the purification of synthesized RNA >25 nucleotides, available in three different binding capacities for flexibility. For removing and inactivating proteins, phenol:chloroform extraction followed by ethanol precipitation can be carried out, though this method will not completely remove free nucleotides. For purification of RNA’s that bind protein tightly, combining phenol:chloroform extraction with spin column purification can be an effective method to get higher recovery of purified RNA free from nucleotides, salts and proteins.