Automation of Viral RNA Extraction from Saliva Using the Monarch RNA Cleanup Kit


The following protocols are for the extraction of total RNA from saliva using the Monarch RNA Cleanup Kit (50 µg) (NEB #T2040) and the QIAcube® or KingFisher® Flex automation platforms. The protocols can also be used with the Monarch RNA Cleanup Columns (NEB #T2047) and associated buffers (NEB #T2041 and #T2042). Note that this protocol does not remove residual DNA and is suitable for RNA-specific downstream methods only (e.g., RT-qPCR or RT-LAMP). We do not recommend on-column DNase I treatment with the Monarch RNA Cleanup Kit as it can often result in residual DNase I activity in eluates. Therefore, if contaminating DNA will interfere with downstream applications, we recommend following this RNA extraction protocol with an in-solution treatment with DNase I (NEB #M0303) and additional RNA cleanup using one of the Monarch RNA Cleanup Kits.

These protocols can also be used for non-viral extraction. In this case, a viral inactivation step with Monarch DNA/RNA Protection Reagent (NEB #T2011) is not necessary.

Viral RNA Extraction from Saliva Using the Monarch RNA Cleanup Kits and the QIAcube

Before You Begin:

  • Reagents/materials supplied by user: ≥95% ethanol, RNase-free microfuge tubes, QIAcube plastics, nuclease-free water.
  • In addition to the Monarch RNA Cleanup Kit, 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.

Protocol:

  1. In a 2 ml QIAcube RB sample tube, add 100 µl (1 volume) of 2X Monarch DNA/RNA Protection Reagent to your 100 µl saliva sample.

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

  3. Run samples on the QIAcube using the RNeasy® QIAshredder Protocol for animal cells with the following setup and 50 µl elution in nuclease-free water:
Rotor Adaptor Positions:
Position Column/Tube Cap Position
1 Monarch RNA Cleanup Column (NEB #T2047) L1
2 Monarch RNA Cleanup Column (NEB #T2047)* *
3 1.5 ml collection tube** L3

* The cap of the second RNA Cleanup Column should be cut off prior to loading into the QIAcube rotor adaptor.
**Sarstedt, Microtube 1.5 ml, as indicated in the QIAcube protocol.

Reagent Positions:
Position Reagent
1 -
2 100% Ethanol
3 -
4 RNA Cleanup Wash Buffer
5 RNA Cleanup Wash Buffer
6 Nuclease-free Water

Viral RNA Extraction from Saliva Using the Monarch Buffers and the KingFisher Flex

Before You Begin:

  • Reagents/materials supplied by user: ≥95% ethanol, RNase-free microfuge tubes, KingFisher Flex plastics, Dynabeads® MyOne Silane magnetic beads
  • 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.

Protocol:

  1. Add 50 µl (1 volume) of 2X Monarch DNA/RNA Protection Reagent to your 50 µl saliva sample and mix by pipetting or vortexing.

  2. Add 200 µl (2 volumes) of RNA Cleanup Binding Buffer and mix by pipetting or vortexing.

  3. Add 300 µl (1 volume) of ethanol and mix by pipetting or vortexing.

  4. Add 20 µl Dynabeads MyOne Silane magnetic beads and vortex to mix.

  5. Transfer samples to a KingFisher Flex compatible 96 deep-well plate.

  6. Agitate the plate at 1000 rpm for 30 min at room temperature.

  7. Steps for the KingFisher Flex:

  8. Prepare wash plate 1 (96 Deep well plate) by aliquoting 500 µl Monarch RNA Cleanup Wash Buffer per well.

  9. Prepare wash plate 2 (96 Deep well plate) by aliquoting 500 µl Monarch RNA Cleanup Wash Buffer per well.

  10. Prepare the elution plate (96 Standard plate) by aliquoting 50 µl Monarch nuclease-free water per well.

  11. Load sample, wash, and elution plates onto the KingFisher Flex worktable for processing.

  12. Process samples using a modified MagMAX Pathogen RNA/DNA (High Volume) protocol with two wash steps, instead of four [i.e., remove two wash steps from the MagMAX Pathogen RNA/DNA (High Volume) protocol]. Place eluted RNA on ice if being used for downstream steps, at -20°C for short-term storage (less than 1 week), or at -80°C for long-term storage. Addition of EDTA to 0.1–1.0 mM may reduce the activity of any contaminating RNases.

*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.