FAQ: Do you have a protocol for separating small and large RNAs into separate fractions?

Yes, the Monarch RNA Cleanup Kit manual contains a protocol which will separate RNA into RNAs > 200 nt and < 200 nt. The protocol can also be accessed online. Following this protocol enriches RNA below 200 nt into the “small RNA” fraction, while RNA above 200 nt is enriched in the “large RNA” fraction. No fractionation protocol allows precise separation of similarly sized RNA based on length alone. We find RNA between 100–200 nt will preferentially partition to the different fractions based on various factors, including predicted secondary structure and interactions with other RNAs within the sample. Highly structured molecules between 100–200 nt typically fall into the small RNA fraction while unstructured RNA between 100–200 nt fall into the large RNA fraction.

The Monarch RNA Cleanup Kit can be used to separate RNA into large and small RNA fractions

HeLa Total RNA (5.2 μg, A1 and B1) was separated into large RNA (A2 and B2) and small RNA (A3 and B3) fractions following the Purification of Small and Large RNAs into Separate Fractions Protocol and running on an Agilent Bioanalyzer® RNA Pico chip. Over 96% (5 μg) of the RNA input was recovered in the separate RNA fractions. Samples 1 and 2 were diluted 1:20 prior to analysis.