Home FAQs What size RNA can be purified with the Monarch RNA Cleanup Kit?

FAQ: What size RNA can be purified with the Monarch RNA Cleanup Kit?

Using the standard protocol, RNAs ≥ 25 nt can be purified. With a slight modification in the protocol (adding 2 volumes of ethanol instead of 1 volume after addition of the binding buffer), RNA > 15 nt can be purified.

Recovery of Small RNAs using the Monarch RNA Cleanup Kit.



Synthesized RNA oligonucleotides (1 μg in 50 μl H2O) of varying lengths (15–100 nt) were purified using the Monarch RNA Cleanup Kit (NEB #T2040) and eluted in 50 μl nuclease-free water. The percent recovery of RNA was calculated from the resulting A260, measured using a Trinean DropSense™ 16. The Monarch RNA Cleanup Kit standard protocol results in >70% recovery and cleanup of RNA ≥ 25 nt, while use of the Monarch RNA Cleanup Kit with the modified protocol (addition of 2 volumes of ethanol in step 2) results in >70% recovery and cleanup of even smaller RNAs (as low as 15 nt).

Please note that recovery of small RNAs (< 45 nt) can be affected by sequence, interactions with other nucleic acids and/or secondary structure.

Secondary structure affects recovery of small RNAs (<45 nt)



Synthesized RNA oligonucleotides (1 μg in 50 μl H2O) of varying lengths (25-40 nt) and varying predicted secondary structure* were purified using the Monarch RNA Cleanup Kit (NEB #T2040) and eluted in 50 μl nuclease-free water. The percent recovery of the RNA was calculated from the resulting A260 as measured using a Trinean DropSense™ 16. Small RNAs (25–40 nt) with low predicted secondary structure were recovered efficiently using the Monarch RNA Cleanup Kit standard protocol. However, increased levels of secondary structure decrease the recovery of small RNAs (25–40 nt in length). Recovery can typically be increased to >70% (orange line) using the Monarch RNA Cleanup Kit with the modified protocol (addition of 2 volumes of ethanol in step 2).

*Secondary structure was predicted using RNAstructure Web Server: Reuter, J. S., & Mathews, D. H. (2010). RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics. 11,129

If poor yield of a small RNA is observed using the standard RNA Cleanup Protocol (adding 1 volume of ethanol prior to binding to the column), we recommend using the modified protocol (adding 2 volumes of ethanol prior to binding to the column), which uses a higher ethanol-to-sample ratio to shift the exclusion cut-off and enable the capture of smaller RNAs.