Home Protocols RNA Extraction from Saliva Using the Monarch RNA Cleanup Kits

RNA Extraction from Saliva Using the Monarch RNA Cleanup Kits


This protocol is for the Monarch RNA Cleanup Kit (NEB #T2040) but can also be used with the Monarch RNA Cleanup Columns (NEB # T2047) and associated buffers (NEB #’s T2041, T2042). If this protocol is being used for viral extraction, an additional step with the Monarch DNA/RNA Protection Reagent (NEB #T2011), which is sold separately, is required.

The standard protocol outlined below will purify RNA ≥ 25 nt. A simple modification in Step 4 can allow for the purification of RNA as small as 15 nt.

For more information, download our technical note, Purification of synthetic SARS-CoV-2 viral RNA from biological samples using the Monarch® Total RNA Miniprep Kit and the Monarch RNA Cleanup Kit.


Before You Begin:

  • For viral extraction: Monarch DNA/RNA Protection Reagent (NEB #T2011) is required (sold separately).
  • This protocol requires the use of 2 RNA Cleanup Columns/sample, effectively reducing the number of preps per S and L kit to 5 and 50, respectively.
  • Add 4 volumes of ethanol (≥ 95%) to one volume of RNA Cleanup Wash Buffer.
  • If a precipitate has formed in the RNA Cleanup Binding Buffer, warm to room temperature to re-dissolve before use.
  • All centrifugation steps should be carried out at room temperature at 16,000 x g (~13,000 RPM).

Protocols:

Viral RNA Extraction from Saliva Samples

  1. Add 100 µl (1 volume) of 2X Monarch DNA/RNA Protection Reagent to a 100 µl saliva sample. A starting sample volume of 100 µl is recommended in order to yield sufficient amounts of RNA. Because of this large starting volume, samples will require reloading the column in Step 4.

  2. Add 400 µl (2 volumes) of RNA Cleanup Binding Buffer.

  3. Insert a column into the collection tube, load the sample onto the first column and close the cap. Spin for 1 minute, then save the flow-through.
    4. To save time, spin for 30 seconds, instead of 1 minute.

  4. Add 600 µl (1 volume) of ethanol (≥ 95%) to the flow-through of the first column and mix by pipetting or flicking the tube. Do not vortex. This will enable the binding of RNA ≥ 25 nt. If you wish to bind RNA as small as 15 nt, add 2 volumes (1200 μl) of ethanol to your sample instead of 1 volume. The addition of 2 volumes of ethanol shifts the cutoff size of RNA binding from 25 nt down to 15 nt.

  5. Insert a second column into a collection tube, load 900 µl of the sample onto the second column and close the cap. Spin for 1 minute, then discard the flow-through. Repeat this step until the entire sample has been loaded onto the spin column.
    6. To save time, spin for 30 seconds, instead of 1 minute.

  6. Re-insert the column into the collection tube. Add 500 µl RNA Cleanup Wash Buffer, spin for 1 minute, the discard the flow-through.
    7. To save time, spin for 30 seconds, instead of 1 minute.

  7. Repeat wash (Step 6).

  8. Transfer column 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.

  9. Add 20-100 µl of nuclease-free water and spin for 1 minute to elute.  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.
    10. To save time, spin for 30 seconds, instead of 1 minute.

*Note: Though NEB has not internally evaluated viral inactivation, users of our products have confirmed that the Monarch DNA/RNA Protection Reagent (NEB #T2011) is effective at inactivating live SARS-CoV-2 virus under certain conditions. Monarch kits are sold for research use only (RUO) and users should always adhere to the safety guidelines of their institution.


RNA Extraction from Saliva Samples

  1. Starting with a 100 µl saliva sample, add 200 µl (2 volumes) of RNA Cleanup Binding Buffer.

  2. Insert a column into the collection tube, load the sample onto the first column and close the cap. Spin for 1 minute, then save the flow-through.
    3. To save time, spin for 30 seconds, instead of 1 minute.

  3. Add 300 µl (1 volume) of ethanol (≥ 95%) to the flow-through of the first column and mix by pipetting or flicking the tube. Do not vortex. This will enable the binding of RNA ≥ 25 nt. If you wish to bind RNA as small as 15 nt, add 2 volumes (600 μl) of ethanol to your sample instead of 1 volume. The addition of 2 volumes of ethanol shifts the cutoff size of RNA binding from 25 nt down to 15 nt.

  4. Insert a second column into a collection tube, load the sample onto the second column and close the cap. Spin for 1 minute, then discard the flow-through.
    5. To save time, spin for 30 seconds, instead of 1 minute.

  5. Re-insert the column into the collection tube. Add 500 µl RNA Cleanup Wash Buffer, spin for 1 minute, the discard the flow-through.
    6. To save time, spin for 30 seconds, instead of 1 minute.

  6. Repeat wash (Step 6).

  7. Transfer column 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.

  8. Add 20-100 µl of nuclease-free water and spin for 1 minute to elute.  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.
    9. To save time, spin for 30 seconds, instead of 1 minute.