Home Protocols Purification of RNA from the Aqueous Phase Following TRIzol®/Chloroform Extraction using the Monarch® RNA Cleanup Kits

Purification of RNA from the Aqueous Phase Following TRIzol®/Chloroform Extraction using the Monarch® RNA Cleanup Kits


RNA isolation reagents containing guanidine thiocyanate and phenol (e.g., TRIzol, RNAzol®, QIAzol®, etc) combined with chloroform extraction, are often used for sample lysis and RNA purification. The aqueous phase from any guanidinium thiocyanate-phenol-chloroform extraction can be cleaned up using the Monarch RNA Cleanup Kits (NEB #T2030, T2040, T2050), thereby eliminating the need for tedious RNA precipitation steps.

Before You Begin:

  • Add 4 volumes of ethanol (≥ 95%) to the Monarch RNA Wash Buffer before use, as directed on the bottle.
  • All centrifugation steps should be carried out at 16,000 x g. (~13K RPM in a typical microcentrifuge). This ensures all traces of buffer are eluted at each step.

Protocol

  1. Following guanidinium-thiocyanate-phenol-chloroform extraction, carefully transfer the upper aqueous phase into an RNase-free tube (not provided).

  2. Add 1 volume of ethanol (≥ 95%). Mix well by pipetting up and down or flicking the tube. Do not vortex.

  3. Insert an RNA cleanup column into a collection tube, load sample onto the column and close the cap. Spin for 1 minute, then discard flow-through. For diluted samples ≥ 900 μl, load a portion of the sample, spin, and then repeat as necessary.

     To save time, spin for 30 seconds, instead of 1 minute.

  4. Re-insert the column into the collection tube. Add 500 μl RNA Cleanup Wash Buffer and spin for 1 minute. Discard the flow-through.

     To save time, spin for 30 seconds, instead of 1 minute.

  5. Repeat wash (Step 4).

  6. Transfer the column to an RNase-free 1.5 ml microfuge tube (not provided). Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to next step. 

  7. Elute in nuclease-free water according to the table below. The eluted RNA can be used immediately or stored at -70ºC. Care should be used to ensure the elution buffer is delivered onto the center of the matrix and not the wall of the column to maximize elution efficiency.

    KIT ELUTION VOLUME INCUBATION TIME SPIN TIME
    T2030 6–20 µl N/A 1 minute
    T2040 20–100 µl N/A 1 minute
    T2050** 50–100 µl 5 minutes (Room temp.) 1 minute

    * When cleaning up large amounts of RNA (> 100 μg, NEB #T2050), some precipitation may occur following the addition of the Monarch RNA Cleanup Binding Buffer and ethanol to the sample (Steps 1 and 2). A pellet containing the RNA of interest may form on the side of the column following the first binding spin (Step 3). To maximize recovery of this RNA, a second elution is recommended.

    ** Yield may slightly increase if a larger volume is used, but the RNA will be less concentrated.

     To save time, spin for 30 seconds, instead of 1 minute.


Recovery of RNA from Monarch RNA Cleanup Kits with Varying Elution Volumes



rRNA (10, 50 or 500 µg, respectively of 16S and 23S Ribosomal Standard from E. coli, Sigma) was purified using a Monarch RNA Cleanup Kit (10 µg, NEB #T2030) (50 µg, NEB #T2040) (500 µg, NEB #T2050). Nuclease-free water was used to elute the RNA. The percent recovery of the RNA was calculated from the resulting A260 as measured using a Trinean DropSense 16. ~80% of RNA can be efficiently recovered in 6 µl from the Monarch RNA Cleanup Kit (10 µg, NEB #T2030), 20 µl from the Monarch RNA Cleanup Kit (50 µg, NEB #T2040), and 50 µl from the Monarch RNA Cleanup Kit (500 µg, NEB #T2050).

 

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