May 04, 2020

Public workspaceRNA Purification from Buccal Swabs, Nasopharyngeal Samples (swab or aspirate) and Saliva using the Monarch Total RNA Miniprep Kit V.1

DOI
dx.doi.org/10.17504/protocols.io.be93jh8n
  • 1New England Biolabs
  • New England Biolabs (NEB)
  • Coronavirus Method Development Community
Isabel Gautreau
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DOI: dx.doi.org/10.17504/protocols.io.be93jh8n
External link: https://www.neb.com/protocols/2020/03/11/rna-purification-from-buccal-swabs-using-the-monarch-total-rna-miniprep-kit-neb-t2010
Protocol Citation: New England Biolabs 2020. RNA Purification from Buccal Swabs, Nasopharyngeal Samples (swab or aspirate) and Saliva using the Monarch Total RNA Miniprep Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.be93jh8n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: April 17, 2020
Last Modified: May 14, 2021
Protocol Integer ID: 35867
Keywords: buccal swab, saliva swab, nasopharyngeal, Viral RNA, RNA Extraction, Saliva RNA, Buccal Swab, Nasopharyngeal swab, viral rna isolation, viral RNA extraction
Abstract
This protocol utilizes the Monarch Total RNA Miniprep Kit to purify RNA from buccal swabs, nasopharyngeal samples, and saliva.
Guidelines
This protocol is to be used for research use only.
Materials
MATERIALS
ReagentNuclease-free Water
ReagentMicrocentrifuge
ReagentMonarch Total RNA Miniprep KitNew England BiolabsCatalog #T2010S
ReagentRNase-free Microfuge Tubes (0.5 mL)Thermo FisherCatalog #AM12300
ReagentRNase-free Microfuge Tubes (1.5 mL)Thermo FisherCatalog #AM12400
Additional Materials:
  • isopropanol
  • ≥95% ethanol
  • 2X Monarch DNA/RNA Protection Reagent
  • collection tubes (additional)
  • Monarch Proteinase K
  • Monarch Lysis Buffer
  • 1X Monarch DNA/RNA Protection Reagent
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
  • 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)
  • 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
  • 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.
Sample Disruption and Homogenization


Step case

Buccal + nasopharyngeal with transport medium
1 step

Buccal swabs and nasopharyngeal samples with transport medium
Place swab into a tube containing Amount300 µL of 1X Monarch DNA/RNA Protection Reagent to an aliquot of transport medium and vortex briefly.
For every Amount300 µL of DNA/RNA Protection Reagent/Sample Mixture , add Amount15 µL Monarch Proteinase K.
Vortex briefly and incubate at TemperatureRoom temperature forDuration00:30:00 .
Incubation
Vortex sample briefly and spin for Duration00:02:00 (16,000 x g) to pellet debris.
Centrifigation
Transfer supernatant to an RNase-free microfuge tube.
Add an equal volume of Monarch RNA Lysis Buffer and vortex briefly.
Proceed to Step 1 of Part 2: RNA Binding and Elution
Part 2: RNA Binding and Elution
Part 2: RNA Binding and Elution

Note
All centrifugation steps should be carried out at 16,000 x g.
Transfer up to Amount800 µL of the sample from Part 1 to a gDNA Removal Column (light blue) fitted with a collection tube.
Note
For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.

Spin for Duration00:00:30 to remove most of the gDNA.
Note
SAVE THE FLOW-THROUGH (RNA partitions here).



Centrifigation
Discard the gDNA Removal Column.
Add an equal volume of ethanol (≥ 95%) to the flow-through and mix throughly by pipetting.
Note
To exclude RNA ≤ 200 nt, add only 1/2 volume ethanol to flow-through. The addition of ethanol creates favorable conditions for RNA to bind to the RNA Purification column.

Mix
Transfer mixture to an RNA Purification Column (dark blue) fitted with a collection tube.
Spin for Duration00:00:30 .
Centrifigation
Discard flow-through.
Note
If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, Step 10.1–10.3 (recommended). If not, proceed to Step 5.

Add Amount500 µL RNA Wash Buffer and spin for Duration00:00:30 and discard flow-through.
Note
This ensures all salts are removed prior to the addition of DNase I.

Note
If using a vacuum manifold, add 500 μl of RNA Wash Buffer and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.


Centrifigation
In an RNase-free microfuge tube (not included), combine Amount5 µL DNase I with Amount75 µL DNase I Reaction Buffer r and pipet mixture directly to the top of the matrix.
Pipetting
Incubate for Duration00:15:00 atTemperatureRoom temperature .
Incubation
Add Amount500 µL RNA Priming Buffer and spin for Duration00:00:30 .
Centrifigation
Discard flow-through.
Note
If using a vacuum manifold, add 500 μl of RNA Priming Buffer and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.

Add Amount500 µL RNA Wash Buffer and spin for Duration00:00:30 .
Centrifigation
Discard flow-through.
Note
If using a vacuum manifold, add 500 μl of RNA Priming Buffer and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.

Add another Amount500 µL RNA Wash Buffer and spin for Duration00:02:00 .
Centrifigation
Transfer column to an RNase-free microfuge tube.
Note
Use care to ensure the tip of the column does not contact the flow-through. If in doubt, re-spin for 1 minute to ensure no ethanol is carried over.

Note
If using a vacuum manifold, add 500 μl of RNA Wash Buffer and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.

Add Amount30 µL toAmount100 µL Nuclease-free Water directly to the center of the column matrix and spin for Duration00:00:30 .
Note
For best results, elute with at least 50 µl, which is the minimum volume needed to wet the membrane. Lower volumes can be used but will result in lower recovery (elution in 30 µl results in > 80% recovery and 100 µl provides maximum recovery). For spectrophotometric analysis of eluted RNA, it may be necessary to re-spin eluted samples and pipet aliquot from top of the liquid to ensure that the A 260/230 is unaffected by possible elution of silica particles.

Centrifigation
Place RNA on ice if being used for downstream steps at:

Temperature-20 °C short-term storage (less than one week)
Temperature-80 °C long-term storage .
Note
Addition of EDTA to 0.1–1.0 mM may reduce the activity of any contaminating RNases.