Total RNA Purification from Mammalian Whole Blood Samples 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:

Mammalian whole blood contains little RNA and an abundance of protein (hemoglobin). Effective lysis can be achieved with Monarch DNA/RNA Protection Reagent. Proteinase K treatment is necessary to lower hemoglobin levels prior to loading the column.

 

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. When processing blood samples, the 2X concentrate is used (without dilution). 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.
  • Note: the gDNA Removal column is not used for whole blood samples.

 

 

Protocol Part 1: Sample Disruption and Homogenization (Fresh or Frozen Mammalian Whole Blood)

  1. Add an equal volume (up to 200 μl) of DNA/RNA Protection Reagent (2X concentrate) to an aliquot of whole blood and vortex briefly. Do not place samples on ice. For frozen samples, quickly thaw in the presence of 2X DNA/RNA Protection Reagent while vortexing or shaking. Blood cells are lysed during this step, releasing the RNA.

  2. For every 400 μl of DNA/RNA Protection Reagent/blood mixture, add 10 μl of Proteinase K (Prot K). Vortex briefly and incubate at room temperature for 30 minutes.

  3. Add an equal volume of isopropanol (not included) and vortex briefly. Addition of isopropanol is necessary to create favorable binding conditions before application onto the RNA Purification Column. Up to 3 ml blood may be processed on a single column with reloading by repeating steps 1-3 with 200 μl of blood at a time.

 

Protocol Part 2: RNA Binding and Elution (Fresh or Frozen Mammalian Whole Blood)

All centrifugation steps should be performed at 16,000 x g.
For sample volumes > 800 μl (column reservoir capacity), columns may be reloaded.

  1. 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 2A–2C (recommended). If not, proceed to Step 3.

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

    2A. 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.

    2B. 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.

    2C. Incubate for 15 minutes at room temperature.


  3. 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.


  4. 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.


  5. 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.


  6. 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.

  7. 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

Guidelines for RNA Extraction and Purification from Whole Blood Samples

Troubleshooting Guide for RNA Purification

Product Manual