Separation of Large and Small RNA into Fractions using the Monarch® RNA Cleanup Kits

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.

Before You Begin:

  • Add 4 volumes of ethanol (≥ 95%) to the Monarch RNA Wash Buffer before use, as directed on the bottle.

  • All centrifugation steps should be carried out at 16,000 x g. (~13K RPM in a typical microcentrifuge). This ensures all traces of buffer are eluted at each step.

  1. A starting sample volume of 50 μl is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. For samples larger than 50 μl, adjust volumes accordingly.

  2. Add 50 μl (1 volume) RNA Cleanup Binding Buffer to the 50 μl sample.

  3. Add 50 μl (1/2 volume) ethanol (≥ 95%). Mix well by pipetting up and down or flicking the tube. Do not vortex.

  4. Insert an RNA Cleanup Column into a collection tube and load sample onto column (Column #1, labeled "large RNAs") and close the cap. Spin for 1 minute. Save the flow through! For samples with a starting volume larger than 300 μl, load a portion of the sample, spin, and then repeat as necessary.

    Small RNAs are in the flow through; to isolate small RNAs, continue with Step 5. Large RNAs are bound to column #1. To isolate large RNAs, continue with Step 7.

  5. Add 150 μl (1 volume) ethanol (≥ 95%) to the flow through and mix.

  6. Transfer the mixture to a new column (Column #2, labeled "Small RNAs") and centrifuge for 1 minute. Discard the flow through.

  7. Insert columns into collection tubes. Add 500 μl RNA Cleanup Wash Buffer to each and spin for 1 minute. Discard the flow-through.

  8. Repeat wash (Step 7) for both columns.

  9. Transfer columns to an RNase-free 1.5 ml microfuge tube (not provided). Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to next step. 

  10. Elute in nuclease-free water according to the table below. The eluted RNA can be used immediately or stored at -70°C. Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency.

    T2030 6–20 µl N/A 1 minute
    T2040 20–100 µl N/A 1 minute
    T2050** 50–100 µl 5 minutes (Room temp.) 1 minute

    * When cleaning up large amounts of RNA (> 100 μg, NEB #T2050), some precipitation may occur following the addition of the Monarch RNA Cleanup Binding Buffer and ethanol to the sample (Steps 1 and 2). A pellet containing the RNA of interest may form on the side of the column following the first binding spin (Step 3). To maximize recovery of this RNA, a second elution is recommended.

    ** Yield may slightly increase if a larger volume is used, but the RNA will be less concentrated.

      To save time, spin for 30 seconds, instead of 1 minute.


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.


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