RNA Purification from Buccal Swabs, Nasopharyngeal Samples (swab or aspirate) and Saliva using the Monarch Total RNA Miniprep Kit (NEB #T2010)

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This protocol has been validated with buccal swabs and saliva samples. We have also confirmed the compatibility of this workflow with viral transport medium using reference samples that do not require cell and/or viral envelope lysis. At this time, we have not yet internally validated this kit with nasopharyngeal samples.


Materials and Equipment

  • Required equipment: microcentrifuge
  • Reagents supplied by user: ≥95% ethanol, RNase-free microfuge tubes, isopropanol
  • Additional equipment/reagents that may be required: nuclease-free water, additional collection tubes

Protocol

Buffer Preparation and Notes Before You Begin:


  • 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.

Protocol Part 1: Sample Disruption and Homogenization

Buccal swabs and nasopharyngeal swabs in transport medium:

  1. Add an equal volume (up to 200 μl) of 2X Monarch DNA/RNA Protection Reagent to an aliquot of transport medium and vortex briefly.
  2. For every 400 μl of DNA/RNA Protection Reagent/Sample mixture add 20 μl Monarch Proteinase K.
  3. Vortex briefly and incubate at room temperature for 30 minutes.
  4. Add an equal volume of Monarch RNA Lysis Buffer and vortex briefly. Proceed to Step 1 of Part 2: RNA Binding and Elution.

Buccal swabs and nasopharyngeal samples without transport medium:

  1. Place swab into a tube containing 300 ul 1X Monarch DNA/RNA Protection Reagent. Vigorously swirl the swab to resuspend the sample material in Protection Reagent.
  2. For every 300 μl of DNA/RNA Protection Reagent/Sample mixture, add 15 μl Monarch Proteinase K. Vortex briefly and incubate at room temperature for 30 minutes.
  3. Vortex sample briefly and spin for 2 min (16,000 x g) to pellet debris. Transfer supernatant to an RNase-free microfuge tube (not included).
  4. Add an equal volume of Monarch RNA Lysis Buffer and vortex briefly. Proceed to Step 1 of Part 2: RNA Binding and Elution.

Saliva samples:

  1. Immediately stabilize 200µl of the saliva sample after collection by mixing with an equal volume of 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. For every 400 μl of DNA/RNA Protection Reagent/Sample mixture add 20 μl Monarch Proteinase K.
  3. Vortex briefly and incubate at room temperature for 30 minutes.
  4. Add an equal volume of Monarch RNA Lysis Buffer, vortex briefly and proceed to Step 1 of Part 2: RNA Binding and Elution.

Protocol Part 2: RNA Binding and Elution

All centrifugation steps should be carried out at 16,000 x g.

  1. Transfer up to 800 μl of the sample from PART 1 to a gDNA Removal Column   (light blue) fitted with a collection tube. For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.

  2. Spin for 30 seconds to remove most of the gDNA. SAVE THE FLOW-THROUGH (RNA partitions here). Discard the gDNA Removal Column.

  3. Add an equal volume of ethanol (≥ 95%) (not included) to the flow-through and mix thoroughly by pipetting. Do not vortex. 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.

  4. Transfer mixture to an RNA Purification Column   (dark blue) fitted with a collection tube. Spin for 30 seconds. Discard flow-through. If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, Step 4A–4C (recommended). If not, proceed to Step 5.

    Optional (but recommended): On-column DNase I treatment for enzymatic removal of residual gDNA

    4A. Add 500 μl RNA Wash Buffer and spin for 30 seconds. Discard flow-through. This ensures all salts are removed prior to the addition of DNase I.
     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.

    4B. In an RNase-free microfuge tube (not included), combine 5 μl DNase I with 75 μl DNase I Reaction Buffer and pipet mixture directly to the top of the matrix.

    4C. Incubate for 15 minutes at room temperature.

  5. Add 500 μl RNA Priming Buffer and spin for 30 seconds. Discard flow-through.  
     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.

  6. Add 500 μl RNA Wash Buffer and spin for 30 seconds. Discard flow-through.
     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.

  7. Add another 500 μl RNA Wash Buffer and spin for 2 MINUTES. Transfer column to an RNase-free microfuge tube (not included). 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.
     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.

  8. Add 30-100 µl Nuclease-free Water directly to the center of column matrix and spin for 30 seconds. 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.

  9. 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.

Additional Resources:

General Guidelines for Successful RNA Purification Using the Monarch Total RNA Miniprep Kit
Guidance on Choosing Sample Input Amounts when Using the Monarch Total RNA Miniprep Kit
Troubleshooting Guide for RNA Purification
Product Manual