RNA Extraction from Buccal/Nasopharyngeal Swabs Using the Monarch RNA Cleanup Kits


This protocol is for the Monarch RNA Cleanup Kits (NEB #T2030 and #T2040) but can also be used with the Monarch RNA Cleanup Columns (NEB #T2037 and #T2047) and associated buffers (NEB #T2041 and #T2042). If this protocol is being used for viral extraction, the Monarch DNA/RNA Protection Reagent (NEB #T2011), which is sold separately, is required for viral inactivation*.

Before You Begin:

  • For viral extraction:  Monarch DNA/RNA Protection Reagent (NEB #T2011)  is required (sold separately).
  • Starting sample can be in either Universal Transport Medium (UTM) or 1X Monarch DNA/RNA Protection Reagent (see different protocols below).
  • Add 4 volumes of ethanol (≥ 95%) to 1 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).

Protocol:

Viral Extraction from Samples in Transport Medium:

The standard protocol outlined below will purify RNA ≥ 25 nt. A simple modification in Step 3 can allow for the purification of RNA as small as 15 nt.

  1. Add 300 µl (1 volume) of 2X Monarch DNA/RNA Protection Reagent to your 300 µl sample in UTM. A starting sample volume of 300 µl is recommended in order to yield sufficient amounts of RNA. Because of this large starting volume, samples will require reloading of the column in Step 4.
  2. Add 1200 µl (2 volumes) of RNA Cleanup Binding Buffer.
  3. Add 1800 µl (1 volume) of ethanol (≥ 95%) 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 (3600 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 the column into the collection tube. Load 900 µl of the sample onto the 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.
  5. 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, then discard the flow-through.
  7. To save time, spin for 30 seconds, instead of 1 minute.
  8. Repeat wash (Step 5).
  9. 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.
  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.
  11. To save time, spin for 30 seconds, instead of 1 minute.

KIT

ELUTION VOLUME

INCUBATION TIME

SPIN TIME

T2030

6–20 µl

N/A

1 minute

T2040

20–100 µl

N/A

1 minute

Extraction from Samples in Transport Medium:

The standard protocol outlined below will purify RNA ≥ 25 nt. A simple modification in Step 2 can allow for the purification of RNA as small as 15 nt.

  1. Starting with a 300 ul sample, add 600 µl (2 volumes) of RNA Cleanup Binding Buffer.
  2. Add 900 µl (1 volume) of ethanol ( 95%) 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 (1800 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.
  3. Insert the column into the collection tube. Load 900 µl of the sample onto the column andclose 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.
  4. 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, then discard the flow-through.
  6. To save time, spin for 30 seconds, instead of 1 minute.
  7. Repeat wash (Step 5).
  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. 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.
  10. To save time, spin for 30 seconds, instead of 1 minute.

KIT

ELUTION VOLUME

INCUBATION TIME

SPIN TIME

T2030

6–20 µl

N/A

1 minute

T2040

20–100 µl

N/A

1 minute

Extraction from Samples Stored in 1X Monarch DNA/RNA Protection Reagent:

Samples stored in Monarch DNA/RNA Protection Reagent do not require an additional viral inactivation step. The standard protocol outlined below will purify RNA ≥ 25 nt. A simple modification in Step 2 can allow for the purification of RNA as small as 15 nt.

  1. Add 600 µl (2 volumes) of RNA Cleanup Binding Buffer to a 300 µl sample in 1X DNA/RNA Protection Reagent. A starting sample volume of 300 µl is recommended in order to yield sufficient amounts of RNA. Because of this large starting volume, samples should be prepared in at least a 2 mL vessel and samples will require reloading the column in Step 3.
  2. Add 900 µl (1 volume) of ethanol (≥ 95%) 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 (1800 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.
  3. Insert the column into the collection tube. Load 900 µl of the sample onto the 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.
  4. 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.
  7. Repeat wash (Step 4).
  8. Transfer the 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. 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.
  10. To save time, spin for 30 seconds, instead of 1 minute.

KIT

ELUTION VOLUME

INCUBATION TIME

SPIN TIME

T2030

6–20 µl

N/A

1 minute

T2040

20–100 µl

N/A

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.