FFPE Tissue Deparaffinization and Subsequent RNA Purification Using the Monarch Total RNA Miniprep Kit  (NEB #T2010)

Materials and Equipment

  • Required equipment: microcentrifuge, water bath or heat blocks (37°C, 55°C and 65°C)
  • Reagents supplied by user: ≥ 95% ethanol, RNase-free microfuge tubes, Xylene or similar for deparaffinization of FFPE tissue
  • Additional equipment/reagents that may be required: nuclease-free water, additional collection tubes

Buffer Preparation and Notes Before You Begin:

  • 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.
  • Paraffin is removed from FFPE samples by treating with xylene or a commercially available FFPE deparaffinization solution (see manufacturer’s recommendations for details).

Rapid Deparaffinization with Xylene (tissue sections)

  1. Remove (trim) as much excess paraffin from the sample as possible and transfer to 1.5 ml tube (not included). 

  2. Add 1 ml xylene (not included) and vortex vigorously 30 seconds. 

  3. Centrifuge for 1 minute at 16,000 x g and remove xylene. 

  4. Wash sample with 1 ml ethanol (≥ 95%) (not included) and vortex vigorously 30 seconds.

  5. Centrifuge for 1 minute at 16,000 x g and remove ethanol. Repeat Steps 4 and 5. 

  6. Dry samples by vacuum centrifugation (e.g. Speed-Vac) or by incubating uncapped tubes at ≤ 37°C for up to 40 minutes. 

  7. Proceed to Tissue Digestion.

Tissue Digestion

  1. Prepare a mix of the following:

    DNase/RNase-Free Water 170 μl
     Proteinase K Reaction Buffer  20 μl
     Proteinase K 10 μl
  2. Add the mixture to the deparaffinized tissue sample (≤ 25 mg).

  3. Incubate at 55°C for 15 minutes.

  4. Transfer the tube to a preheated block at 65°C and incubate for 15 minutes. Note: If only one heat block is available, place sample at room temperature after the 55°C incubation until heating block has reached 65°C.

  5. Add 600 μl RNA Lysis Buffer and mix thoroughly. Centrifuge for 1 min at 16,000 x g to pellet debris. Transfer supernatant to an RNase-free microfuge tube (not included). Proceed to RNA Binding and Elution.

RNA Binding and Elution

  1. Transfer up to 800 μl of the sample to a gDNA Removal Column symbol_lightbluebullet (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 symbol_bluebullet (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 5). If not, proceed to step 6.

  5. Optional (but recommended): On-column DNase I treatment for enzymatic removal of residual gDNA
    5A. 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.

    5B. 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. 

    5C. Incubate for 15 minutes at room temperature. 

  6. 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.

  7. 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.

  8. 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. 

  9. 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.

  10. 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.