Total RNA Purification from Tough-to-Lyse Samples (bacteria, yeast, plant, etc.) using the Monarch Total RNA Miniprep Kit (NEB #T2010)

Below is a detailed protocol containing explanations and commentary. If you prefer a more concise protocol, please use our Quick Protocol or download our Quick Protocol Card.

Considerations for Sample Disruption and Homogenization:

These samples benefit from mechanical disruption and homogenization to effectively release RNA due to carbohydrate-containing cell walls. Alternatively, samples may be lysed enzymatically by the addition of glycanases (i.e. lysozyme, Zymolyase). Cells grown in liquid culture should be pelleted and separated from culture medium prior to sample disruption and homogenization. These samples benefit from mechanical disruption and homogenization to effectively release RNA due to carbohydrate-containing cell walls. Alternatively, samples may be lysed enzymatically by the addition of glycanases. Both protocols are included below. 

Materials and Equipment

• Required equipment: microcentrifuge, water bath or heat block (55°C)
• Reagents supplied by user: ≥ 95% ethanol, RNase-free microfuge tubes
• Additional equipment/reagents that may be required: nuclease-free water, additional collection tubes, homogenizer (bead mill or other), glycanases for bacteria and yeast

Protocol

Buffer Preparation and Notes Before You Begin:

• Monarch DNA/RNA Protection Reagent is supplied as a 2X concentrate. Dilute 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 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 (Tough-to-Lyse Samples, including bacteria, yeast, plant, etc.)

Mechanical lysis/homogenization is recommended for tough-to-lyse samples. Alternatively, bacteria and yeast may be lysed enzymatically. Some gram-negative bacteria (e.g., E. coli ) do not require mechanical or enzymatic lysis and can by lysed directly by resuspension in DNA/RNA Protection Reagent, however, yields may be lower.

Mechanical Lysis:

1. Determine the amount of 1X DNA/RNA Protection Reagent that you will need according to the table below. Prepare the aliquot of 1X DNA/RNA Protection Reagent by diluting the 2X concentrate with nuclease-free water (not provided for this step).
   
   

   SAMPLE INPUT AMOUNT			VOLUME 1X DNA/RNA PROTECTION REAGENT
   BACTERIA	YEAST	PLANT
   < 5 x 107	< 5 x 106		400 µl
   5 x 107 – 1 x 109	5 x 106 – 5 x 107	≤100 mg	800 µl

2. Add 1X DNA/RNA Protection Reagent to sample.
   
   
3. Disrupt sample mechanically using a bead mill or similar device.
   
   
4. Transfer lysate/homogenate to an RNase-free microfuge tube (not included).
   
   
5. Spin for 2 min (16,000 x g) to pellet debris. Transfer supernatant to an RNase-free microfuge tube (not included).
   
   
6. Add an equal volume of RNA Lysis Buffer and vortex briefly. Proceed to PART 2: RNA Binding and Elution

Enzymatic Lysis of Bacteria or Yeast:

1. Add appropriate reaction buffer and glycanase (not included), and incubate at appropriate temperature according to the chart below:
   
   

   SAMPLE TYPE	ENZYME	INCUBATION TIME	DIGESTION TEMP.
   Bacteria (gram negative)	Lysozyme (1 mg/ml)	5 min	25°C
   Bacteria (gram positive)	Lysozyme (3 mg/ml)	10 min	25°C
   Yeast	Zymolyase (25 U)	30-40 min	30°C

2. Add 2 volumes RNA Lysis Buffer and vortex vigorously for ~10 sec.
   
   
3. Centrifuge for 2 min at 16,000 x g to pellet cellular debris.
   
   
4. Transfer supernatant to an RNase-free microfuge tube (not included). Proceed to PART 2: RNA Binding and Elution

Protocol Part 2: RNA Binding and Elution (Tough-to-Lyse Samples, including bacteria, yeast, plant, etc.)

  All centrifugation steps should be carried out at 16,000 x g.
For sample volumes >800 μl, columns may be reloaded.

For sample volumes >800 µl, columns may be reloaded.

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.

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