Total RNA Purification from Cultured Mammalian Cells 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:

Cells grown in suspension, in a monolayer, or as adherent cells should be pelleted prior to use of this kit to ensure the cell culture medium is removed. The plasma membrane of these samples is easily lysed by the detergents in the lysis buffer and no additional disruption is necessary. Cells grown in suspension, in a monolayer, or as adherent cells should be pelleted prior to use of this kit to ensure cell culture medium is removed. To remove any remaining culture medium, cells may be washed in 1X PBS, pelleted, and then the supernatant can be discarded. The plasma membrane of these samples is easily lysed by detergents in the Lysis Buffer, and no additional disruption is necessary. 

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

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).
   
• Proteinase K is not used for the Cultured Cell protocol, so there is no need to reconstitute this enzyme.
   
• 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 (Cultured Cells) 

1. Pellet cells by centrifugation (500 x g) for 1 minute. Discard supernatant. 
   
   Alternatively, cells in suspension can be directly processed by adding 4 volumes of RNA Lysis Buffer, mixing, and proceeding to Step 1 of PART 2: RNA Binding and Elution. The buffers provided in this kit are optimized for pelleted cells. Lysing cells in suspension often requires large volumes of lysis buffer.
   
   
2. Resuspend pellet in RNA Lysis Buffer (according to table below) by pipetting gently to avoid foaming. Do not place samples on ice. For frozen pellets, thaw briefly before resuspension. For cells previously mixed with DNA/RNA Protection Reagent, add an equal volume of RNA Lysis Buffer.
   
   

   AMOUNT OF CELLS	VOLUME OF RNA LYSIS BUFFER
   up to 3 x 106	300 μl
   3 x 106 to 1 x 107	≥ 600 μl

         

Protocol Part 2: RNA Binding and Elution (Cultured Mammalian Cells)

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