Automation of RNA Extraction from Saliva Using the Monarch Total RNA Miniprep Kit

 

The following protocols are for the extraction of total RNA, including viral RNA, from saliva using the Monarch Total RNA Miniprep Kit (NEB #T2010) and the QIAcube® (Classic model) or KingFisher Flex automation platforms.

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

RNA Extraction from Saliva on the QIAcube

Materials and Equipment

  • ≥95% ethanol
  • RNase-free microfuge tubes
  • QIAcube plastics
  • Monarch DNase I, DNase I Reaction Buffer (NEB #T2019L), nuclease-free water may also be required

Buffer and Reagent Preparation:

  • Monarch DNA/RNA Protection Reagent is supplied as a 2X concentrate. Dilute with nuclease-free water only as needed, as some sample types require resuspension in the 2X concentrate, while others require a 1X solution. If purifying samples stored in Monarch DNA/RNA Protection Reagent, please review the related guidance.
  • For the 50-prep kit, add 275 μl nuclease-free water to the lyophilized DNase I vial and resuspend by gentle inversion. We suggest making aliquots of DNase I, sized to your processing needs, and storing at -20°C to minimize freeze-thaw cycles (3 F/T cycles maximum). The T2010 kit includes DNase I and DNase I Reaction Buffer for 50 manual preps, or 42 QIAcube preps.
  • For the 50-prep kit, add 1,040 μl Proteinase K Resuspension Buffer to the lyophilized Proteinase K (Prot K) vial and vortex to resuspend. Store at -20°C.
  • For the 50-prep kit, add 100 ml ethanol ≥ 95% (not included) to the 25 ml RNA Wash Buffer concentrate and store at room temperature.

Notes Before You Begin:

  • Addition of RNA Lysis Buffer and all subsequent steps should be performed at room temperature (this will prevent precipitation of detergent in the lysis buffer). If samples are accidentally placed on ice and precipitate forms, allow the samples to return to room temperature to resolubilize before loading onto the column.
  • The Quick Saliva Protocol is recommended for the fastest sample processing. This shortened protocol does not utilize DNA/RNA Protection Reagent and Proteinase K.

Protocol

Part 1: Preparing Saliva Samples for QIAcube Processing

Quick Saliva Protocol

  1. To a 200 μl saliva sample, add two volumes (400 μl) Monarch RNA Lysis Buffer, vortex to mix.

  2. Transfer sample to a 2 ml QIAcube RB sample tube.

  3. Proceed to Part 2.

Saliva Protocol with Proteinase K

  1. Immediately stabilize 150 µl of the saliva sample after collection by mixing with an equal volume (150 μl) 2X Monarch DNA/RNA Protection Reagent. If unable to add the Protection Reagent immediately, briefly place saliva sample on ice until Protection Reagent can be added.

  2. Add 15 μl Monarch Proteinase K.

  3. Vortex to mix and incubate at room temperature for 30 minutes.

  4. Add an equal volume (300 μl) Monarch RNA Lysis Buffer and vortex to mix. 

  5. Transfer sample to a 2 ml QIAcube RB sample tube.

  6. Proceed to Part 2.

Part 2: Steps Taken on the QIAcube Worktable

  1. Prepare a DNase I digestion mix in a 2 ml QIAcube RB sample tube; for each sample that will be processed, combine 6 μl Monarch DNase I and 87 μl Monarch DNase I Reaction Buffer. Load the DNase I digestion mix tube into Microcentrifuge Tube Slot A on the worktable. Users may determine that DNase I digestion is optional, in which case the RNeasy Mini QIAshredder protocol for animal cells should be used.

  2. Load samples into the Shaker rack on the QIAcube worktable.

  3. Prepare a Rotor adapter for each sample with columns and an elution tube, as described in the table below. Place loaded rotor adapters into the QIAcube microcentrifuge.

  4. Load reagents in QIAcube bottles into the Reagent Bottle Rack as described in the table below.

  5. Process samples using the RNeasy Mini QIAshredder DNase digest protocol for animal cells, with a 100 μl elution volume.  When processing less than 12 samples, refer to the QIAcube manual for guidance on loading the centrifuge and Shaker rack.

  6. Place 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.

Rotor Adapter

Position Column/Tube Cap Position
1 Monarch RNA Purification Column (NEB #T2007) L1
2 Monarch gDNA Removal Column (NEB #T2017) Cap must be cut off prior to loading
3 Sarstedt 1.5 ml elution tube* L3

*As indicated in the QIAcube protocol

Reagent Bottle Rack

Position Reagent
1 -
2 ≥ 95% Ethanol
3 -
4 Monarch RNA Priming Buffer (NEB #T2013)
5 Monarch RNA Wash Buffer (NEB #T2014)
6 Monarch Nuclease-free Water (NEB #B1500)

Microcentrifuge Tube Slots

Position Content
A DNase I digestion mix
B -
C -

 

RNA Extraction from Saliva on the KingFisher Flex

Materials and Equipment

  • ≥95% ethanol
  • RNase-free microfuge tubes
  • KingFisher Flex plastics
  • Dynabeads® MyOne Silane magnetic beads
  • Nuclease-free water may be required

Buffer and Reagent Preparation:

  • Monarch DNA/RNA Protection Reagent is supplied as a 2X concentrate. Dilute with nuclease-free water only as needed, as some sample types require resuspension in the 2X concentrate, while others require a 1X solution. If purifying samples stored in Monarch DNA/RNA Protection Reagent, please review the related guidance.
  • For the 50-prep kit, add 1,040 μl Proteinase K Resuspension Buffer to the lyophilized Proteinase K (Prot K) vial and vortex to resuspend. Store at -20°C.
  • For the 50-prep kit, add 100 ml ethanol ≥ 95% (not included) to the 25 ml RNA Wash Buffer concentrate and store at room temperature

Notes Before You Begin:

  • Addition of RNA Lysis Buffer and all subsequent steps should be performed at room temperature (this will prevent precipitation of detergent in the lysis buffer). If samples are accidentally placed on ice and precipitate forms, allow the samples to return to room temperature to resolubilize before loading onto the column.
  • DNase I and DNase I Reaction Buffer are not required for this workflow that uses the KingFisher Flex MagMAX Pathogen RNA/DNA (High Volume) protocol.
  • The Quick Saliva Protocol is recommended for the fastest sample processing. This shortened protocol does not utilize DNA/RNA Protection Reagent and Proteinase K.

Protocol

Part 1: Preparing Saliva Samples for Processing on the KingFisher Flex

Quick Saliva Protocol

  1. To 125 µl of the saliva sample, add 2 volumes (250 µl) of Monarch RNA Lysis Buffer and vortex to mix.

  2. Add an equal volume (375 µl) of ethanol, vortex briefly to mix.

  3. Add 20 µl Dynabeads MyOne Silane magnetic beads, vortex to mix.

  4. Transfer samples to a KingFisher Flex compatible 96 Deep well plate.

  5. Agitate the plate at 1000 rpm, 30 min at room temperature. Users may determine that the 30 min agitation step in this Quick Protocol can be shortened or eliminated.

  6. Proceed to Part 2.

Saliva Protocol with Proteinase K:

  1. Immediately stabilize 100 µl of the saliva sample after collection by mixing with an equal volume (100 μl) 2X Monarch DNA/RNA Protection Reagent. If unable to add the Protection Reagent immediately, briefly place saliva sample on ice until Protection Reagent can be added.

  2. Add 10 μl Monarch Proteinase K.

  3. Vortex to mix and incubate at room temperature for 30 minutes.

  4. Add an equal volume (200 μl) Monarch RNA Lysis Buffer and vortex to mix. 

  5. Add an equal volume (400 µl) of ethanol and vortex briefly to mix.

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

  7. Transfer samples to a KingFisher Flex compatible 96 Deep well plate.

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

  9. Proceed to Part 2.

Part 2: Steps Taken on the KingFisher Flex

  1. Prepare Wash plate 1 (96 deep well plate) by aliquoting 500 μl Monarch Priming Buffer per well.

  2. Prepare Wash plates 2 and 3 (96 deep well plates) by aliquoting 500 μl Monarch RNA Wash Buffer per well.

  3. Prepare the elution plate (96 well microplate) by aliquoting 100 μl Monarch nuclease-free water per well.

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

  5. Process samples using a modified MagMAX Pathogen RNA/DNA (High Volume) protocol with three wash steps, instead of four (i.e., remove one wash step from the MagMAX Pathogen RNA/DNA (High Volume) protocol).

  6. Place 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.

RNA Extraction from Saliva on the KingFisher Flex

Plate position 1 2 3 4 5 6

Plate type

96 deep well

96 deep well

96 deep well

96 deep well

96 well microplate

Tip comb in 96 well microplate

Plate identification

Sample plate

Wash plate 1

Wash plate 2

Wash plate 3

Elution plate

n/a

Plate contents

~800 µl Sample lysate +beads

RNA Priming Buffer (500 µl per well)

RNA Wash Buffer (500 µl per well)

RNA Wash Buffer (500 µl per well)

Nuclease-free water (100 µl per well)

n/a

Note: Though NEB has not internally evaluated viral inactivation, users of our products have confirmed that the Monarch DNA/RNA Protection Reagent (NEB #T2011) and Monarch RNA Lysis Buffer (NEB #T2012) are each 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.