Fractionation of Small and Large RNA Using the Monarch Total RNA Miniprep Kit (NEB #T2010)

Materials and Equipment

• Required equipment: microcentrifuge
• 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:

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

Modification of the protocol used for cultured cells can allow for selective depletion or enrichment of the sub-200 nucleotide RNA pool. This modification may also be used on previously purified total RNA to enable fractionation. The procedure is compatible with up to 10⁶ animal cells, or previously isolated RNA (with a sample volume adjusted to ≥ 50 μl).

1. Prepare an equal mixture of RNA Lysis Buffer and ethanol (≥ 95%) (not included). Choose the amount needed based on the number and type of samples. You will need 300 μl of the mixture for each cell pellet, or 2 volumes of the mixture for each RNA aliquot.
   
   
2. Add 300 μl of the mixture to each cell pellet, or 2 volumes of the mixture to each purified RNA aliquot. Mix samples by pipetting.
   
   
3. Transfer the sample into an RNA Purification Column  (dark blue) fitted with a collection tube and spin for 30 seconds at 16,000 x g. SAVE THE FLOW-THROUGH. RNAs > 200 nt bind to the column, whereas RNAs < 200 nt partition into the flow-through.
   
   
4. 4a. To purify RNAs > 200 nt that are bound to the column, proceed to either DNase I treatment (Step 5) or addition of Priming Buffer (Step 6).
   
   4b. To enrich for RNAs < 200 nt, add an equal volume of ethanol (≥ 95%) (not included) to the flow-through from Step 3 and transfer to a new RNA Purification Column fitted with a collection tube. Spin for 30 seconds at 16,000 x g, discard flow-through, and proceed to either DNase I treatment (Step 5) or addition of Priming Buffer (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.

Additional Information 

Guidance on Choosing Sample Input Amounts when using the Monarch Total RNA Miniprep Kit

General Guidelines for Successful RNA Purification using the Monarch Total RNA Miniprep Kit