TITLE: RNA Purification from Buccal Swabs, Nasopharyngeal Samples (swab or aspirate) and Saliva using the Monarch Total RNA Miniprep Kit
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol utilizes the Monarch Total RNA Miniprep Kit to purify RNA from buccal swabs, nasopharyngeal samples, and saliva.</div></div>

[STEPS]
?. [Part 1: Sample Disruption and Homogenization]
Sample Disruption and Homogenization
?. [Part 2: RNA Binding and Elution]
?. [Part 1: Sample Disruption and Homogenization]
Immediately stabilize  after collection by mixing with an equal volume of 2X Monarch DNA/RNA Protection Reagent.
[of the saliva sample]
If unable to add the Protection reagent immediately, briefly place saliva sample on ice until Protection Reagent can be added.
?. [Part 1: Sample Disruption and Homogenization]
For every  , add
[of DNA/RNA Protection Reagent/Sample Mixture]
[Monarch Proteinase K.]
?. [Part 1: Sample Disruption and Homogenization]
Vortex briefly and incubate at  for.
0 Room temperature
?. [Part 1: Sample Disruption and Homogenization]
Add an equal volume of Monarch RNA Lysis Buffer and vortex briefly.
?. [Part 1: Sample Disruption and Homogenization]
?. [Part 1: Sample Disruption and Homogenization]
Add an equal volume (up to ) of 2X Monarch DNA/RNA Protection Reagent to an aliquot of transport medium and vortex briefly.
200 µl
?. [Part 1: Sample Disruption and Homogenization]
For every , add .
[DNA/RNA Protection Reagent/Sample mixture]
[Monarch Proteinase K]
?. [Part 1: Sample Disruption and Homogenization]
Vortex briefly and incubate at  for .
0 Room temperature
?. [Part 1: Sample Disruption and Homogenization]
Add an equal volume of Monarch RNA Lysis Buffer and vortex briefly.
?. [Part 1: Sample Disruption and Homogenization]
Place swab into a tube containing . Vigorously swirl the swab to resuspend the sample material in Protection Reagent.
[1X Monarch DNA/RNA Protection Reagent]
?. [Part 1: Sample Disruption and Homogenization]
For every , add .
[DNA/RNA Protection Reagent/Sample mixture]
[Monarch Proteinase K]
?. [Part 1: Sample Disruption and Homogenization]
Vortex sample briefly and spin for  to pellet debris. Transfer supernatant to an RNase-free microfuge tube (not included).
Centrifuge: 16000 34, 2 min
?. [Part 1: Sample Disruption and Homogenization]
Add an equal volume of Monarch RNA Lysis Buffer and vortex briefly.
?. [Part 1: Sample Disruption and Homogenization]
Immediately stabilize  after collection by mixing with an equal volume of 2X Monarch DNA/RNA Protection Reagent.
[of the saliva sample]
If unable to add the Protection reagent immediately, briefly place saliva sample on ice until Protection Reagent can be added.
?. [Part 1: Sample Disruption and Homogenization]
For every  , add
[of DNA/RNA Protection Reagent/Sample Mixture]
[Monarch Proteinase K.]
?. [Part 1: Sample Disruption and Homogenization]
Vortex briefly and incubate at  for.
0 Room temperature
?. [Part 1: Sample Disruption and Homogenization]
Add an equal volume of Monarch RNA Lysis Buffer and vortex briefly.
?. [Part 1: Sample Disruption and Homogenization]
Proceed to Step 1 of Part 2: RNA Binding and Elution
?. [Part 1: Sample Disruption and Homogenization]
Proceed to Step 1 of Part 2: RNA Binding and Elution.
?. [Part 1: Sample Disruption and Homogenization]
Vortex briefly and incubate at  for .
0 Room temperature
?. [Part 1: Sample Disruption and Homogenization]
Proceed to Step 1 of Part 2: RNA Binding and Elution.
?. [Part 2: RNA Binding and Elution]
Transfer up to  to a gDNA Removal Column (light blue) fitted with a collection tube.
[of the sample from Part 1]
All centrifugation steps should be carried out at 16,000 x g.
For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.
?. [Part 2: RNA Binding and Elution]
Spin for  to remove most of the gDNA.
SAVE THE FLOW-THROUGH (RNA partitions here).
?. [Part 2: RNA Binding and Elution]
Discard the gDNA Removal Column.
?. [Part 2: RNA Binding and Elution]
Add an equal volume of ethanol (≥ 95%) to the flow-through and mix throughly by pipetting.
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.
?. [Part 2: RNA Binding and Elution]
Transfer mixture to an RNA Purification Column (dark blue) fitted with a collection tube.
?. [Part 2: RNA Binding and Elution]
Spin for .
?. [Part 2: RNA Binding and Elution]
Discard flow-through.
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.
?. [Part 2: RNA Binding and Elution]
Add  and spin for  and discard flow-through.
[RNA Wash Buffer]
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.
?. [Part 2: RNA Binding and Elution]
In an RNase-free microfuge tube (not included), combine with  and pipet mixture directly to the top of the matrix.
[DNase I]
[DNase I Reaction Buffer]
?. [Part 2: RNA Binding and Elution]
Incubate for  at.
0 Room temperature
?. [Part 2: RNA Binding and Elution]
Add  and spin for .
[RNA Priming Buffer]
?. [Part 2: RNA Binding and Elution]
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.
?. [Part 2: RNA Binding and Elution]
Add  and spin for .
[RNA Wash Buffer]
?. [Part 2: RNA Binding and Elution]
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.
?. [Part 2: RNA Binding and Elution]
Add another  and spin for .
[RNA Wash Buffer]
?. [Part 2: RNA Binding and Elution]
Transfer column to an RNase-free microfuge tube.
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.
?. [Part 2: RNA Binding and Elution]
Add  to directly to the center of the column matrix and spin for .
30 µl
[Nuclease-free Water]
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.
?. [Part 2: RNA Binding and Elution]
Place RNA on ice if being used for downstream steps at: (less than one week).
[short-term storage]
[long-term storage]
Addition of EDTA to 0.1–1.0 mM may reduce the activity of any contaminating RNases.
?. [Part 1: Sample Disruption and Homogenization]
Place swab into a tube containing  to an aliquot of transport medium and vortex briefly.
[of 1X Monarch DNA/RNA Protection Reagent]
?. [Part 1: Sample Disruption and Homogenization]
For every  , add
[of DNA/RNA Protection Reagent/Sample Mixture]
[Monarch Proteinase K.]
?. [Part 1: Sample Disruption and Homogenization]
Vortex briefly and incubate at  for.
0 Room temperature
?. [Part 1: Sample Disruption and Homogenization]
Vortex sample briefly and spin for  to pellet debris.
[(16,000 x g)]
?. [Part 1: Sample Disruption and Homogenization]
Transfer supernatant to an RNase-free microfuge tube.
?. [Part 1: Sample Disruption and Homogenization]
Add an equal volume of Monarch RNA Lysis Buffer and vortex briefly.
?. [Part 1: Sample Disruption and Homogenization]
Proceed to Step 1 of Part 2: RNA Binding and Elution
?. [Part 1: Sample Disruption and Homogenization]
For every , add .
[of the DNA/RNA Protection Reagent/Sample mixture]
[of Monarch Proteinase K]
?. [Part 1: Sample Disruption and Homogenization]
Add an equal volume (up to  ) of the 2X Monarch DNA/RNA Protection Reagent to an aliquot of transport medium and vortex briefly.
200 µl
?. [Part 1: Sample Disruption and Homogenization]
Vortex briefly and incubate at  for .
0 Room temperature
?. [Part 1: Sample Disruption and Homogenization]
Add an equal volume of Monarch RNA Lysis Buffer and vortex briefly.
?. [Part 1: Sample Disruption and Homogenization]
Proceed to Step 1 of Part 2: RNA Binding and Elution.
?. [Part 2: RNA Binding and Elution]
Transfer up to  to a gDNA Removal Column (light blue) fitted with a collection tube.
[of the sample from Part 1]
All centrifugation steps should be carried out at 16,000 x g.
For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.
?. [Part 2: RNA Binding and Elution]
Spin for  to remove most of the gDNA.
SAVE THE FLOW-THROUGH (RNA partitions here).
?. [Part 2: RNA Binding and Elution]
Discard the gDNA Removal Column.
?. [Part 2: RNA Binding and Elution]
Add an equal volume of ethanol (≥ 95%) to the flow-through and mix throughly by pipetting.
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.
?. [Part 2: RNA Binding and Elution]
Transfer mixture to an RNA Purification Column (dark blue) fitted with a collection tube.
?. [Part 2: RNA Binding and Elution]
Spin for .
?. [Part 2: RNA Binding and Elution]
Discard flow-through.
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.
?. [Part 2: RNA Binding and Elution]
Add  and spin for  and discard flow-through.
[RNA Wash Buffer]
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.
?. [Part 2: RNA Binding and Elution]
In an RNase-free microfuge tube (not included), combine with r and pipet mixture directly to the top of the matrix.
[DNase I]
[DNase I Reaction Buffer]
?. [Part 2: RNA Binding and Elution]
Incubate for  at.
0 Room temperature
?. [Part 2: RNA Binding and Elution]
Add  and spin for .
[RNA Priming Buffer]
?. [Part 2: RNA Binding and Elution]
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.
?. [Part 2: RNA Binding and Elution]
Add  and spin for .
[RNA Wash Buffer]
?. [Part 2: RNA Binding and Elution]
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.
?. [Part 2: RNA Binding and Elution]
Add another  and spin for .
[RNA Wash Buffer]
?. [Part 2: RNA Binding and Elution]
Transfer column to an RNase-free microfuge tube.
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.
?. [Part 2: RNA Binding and Elution]
Add  to directly to the center of the column matrix and spin for .
30 µl
[Nuclease-free Water]
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.
?. [Part 2: RNA Binding and Elution]
Place RNA on ice if being used for downstream steps at: (less than one week).
[short-term storage]
[long-term storage]
Addition of EDTA to 0.1–1.0 mM may reduce the activity of any contaminating RNases.
?. [Part 2: RNA Binding and Elution]
Transfer up to  to a gDNA Removal Column (light blue) fitted with a collection tube.
All centrifugation steps should be carried out at 16,000 x g.
[of the sample from Part 1]
For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.
?. [Part 2: RNA Binding and Elution]
Spin for  to remove most of the gDNA.
SAVE THE FLOW-THROUGH (RNA partitions here).
?. [Part 2: RNA Binding and Elution]
Discard the gDNA Removal Column.
?. [Part 2: RNA Binding and Elution]
Add an equal volume of ethanol (≥ 95%) to the flow-through and mix throughly by pipetting.
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.
?. [Part 2: RNA Binding and Elution]
Transfer mixture to an RNA Purification Column (dark blue) fitted with a collection tube.
?. [Part 2: RNA Binding and Elution]
Spin for .
?. [Part 2: RNA Binding and Elution]
Discard flow-through.
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.
?. [Part 2: RNA Binding and Elution]
Add  and spin for  and discard flow-through.
[RNA Wash Buffer]
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.
?. [Part 2: RNA Binding and Elution]
In an RNase-free microfuge tube (not included), combine with r and pipet mixture directly to the top of the matrix.
[DNase I]
[DNase I Reaction Buffer]
?. [Part 2: RNA Binding and Elution]
Incubate for  at.
0 Room temperature
?. [Part 2: RNA Binding and Elution]
Add  and spin for .
[RNA Priming Buffer]
?. [Part 2: RNA Binding and Elution]
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.
?. [Part 2: RNA Binding and Elution]
Add  and spin for .
[RNA Wash Buffer]
?. [Part 2: RNA Binding and Elution]
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.
?. [Part 2: RNA Binding and Elution]
Add another  and spin for .
[RNA Wash Buffer]
?. [Part 2: RNA Binding and Elution]
Transfer column to an RNase-free microfuge tube.
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.
?. [Part 2: RNA Binding and Elution]
Add  to directly to the center of the column matrix and spin for .
30 µl
[Nuclease-free Water]
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.
?. [Part 2: RNA Binding and Elution]
Place RNA on ice if being used for downstream steps at: (less than one week).
[short-term storage]
[long-term storage]
Addition of EDTA to 0.1–1.0 mM may reduce the activity of any contaminating RNases.