Home Resources General Guidelines for Successful RNA Purification Using the Monarch Spin RNA Isolation Kit (Mini) (NEB #T2110)

General Guidelines for Successful RNA Purification Using the Monarch Spin RNA Isolation Kit (Mini) (NEB #T2110)

Before your Prep:

  1. Work areas should be free of RNase contamination. Wipe bench tops with cleaning agents (i.e., RNaseZap), use dedicated RNase-free pipettes, glass- and plastic-ware, and always wear gloves.
  1. Reconstitute the Monarch Buffer BX, Monarch Buffer WZ and Monarch DNase I, Lyophilized as per the instructions under Reagent & Buffer Preparation Guide.

  2. Ensure that you are using the correct protocol and have all necessary materials and equipment on hand. The protocol consists of Part 1: Sample Lysis and Homogenization, and Part 2: RNA Binding and Elution. Because of the kit’s versatility, the protocol for Part 1 differs according to sample type.

  3. Review the protocol for your sample type before starting. When applicable, pre-heat your heating block to 55°C prior to starting your prep so it will be at the correct temperature for the Proteinase K incubation.

  4. Fresh samples should be rapidly processed after harvest, flash frozen for later use, or stored in a stabilization reagent such as Monarch StabiLyse DNA/RNA Buffer (included in the kit) or other alternatives to protect the RNA integrity in the sample.

  5. Choose input amounts carefully to ensure adequate buffer volumes and columns are not overloaded. Improper input amounts can reduce the RNA's yield, purity, and integrity.

 

During your Prep:

  1. Follow all protocol steps. Optional steps are not necessary but recommended.

  2. Ensure your samples are completely lysed and homogenized to release RNA and maximize yield. Sample types such as some tissues, bacteria, plants, and insects are harder to lyse and benefit from mechanical homogenization using bead beating. If multiple rounds of homogenization are recommended, place samples on ice for one minute between rounds to prevent overheating.

  3. Do not place samples on ice after adding Monarch StabiLyse DNA/RNA Buffer. Adding Monarch StabiLyse DNA/RNA Buffer and all subsequent steps should be performed at room temperature to prevent the precipitation of detergents in buffers.

  4. Label the collection tube fitted to the gDNA removal step and SAVE THE FLOW-THROUGH for this step as the RNA partitions into the flow-through. Since the Monarch Spin Column S2C will be discarded after spinning, it is important to label the collection tube for sample identification. Addition of ethanol to the flow-through creates favorable conditions for RNA to bind to the Monarch Spin Column S2A.

  5. If further gDNA removal is important for downstream applications, be sure to perform a DNase I treatment. This can be done on- or off-column. If performing the on-column DNase I treatment (which is recommended), don’t forget the desalt wash step with Monarch Buffer WZ prior to adding the enzyme. It also helps to prepare a master mix of Monarch DNase I and DNase I Reaction Buffer if you will perform multiple preps simultaneously.

  6. Perform all wash steps. The washes will remove contaminants, including DNase I and salts; it is particularly important to spin the Monarch Spin Column S2A for 2 minutes after the last wash.

  7. Don’t let the tip of the Monarch Spin Column S2A contact the flow-through in the collection tube after the final wash to prevent salt and/or ethanol carry-over. If unsure, repeat centrifugation.

  8. Adjust your elution volume depending on your downstream needs. An elution volume from 20 µl - 100 µl is suggested for maximal recovery. However, elution as low as 10 µl leads to only a minimal loss in recovery.

 

After your Prep:

  1. Spin your sample before taking an aliquot for spectrophotometric analysis.
  1. Place eluted RNA on ice for immediate use or freeze for long-term storage. Avoid unnecessary freeze-thaw cycles of purified RNA. Aliquots should be made consistent with downstream needs.
  1. Elution and storage in nuclease-free water in standard, but the user may add EDTA to the eluted RNA (to 0.1-1 mM) if it will be stored for an extended period. Adding EDTA may protect RNA samples stored for an extended time.

  2. If further gDNA removal is essential for downstream applications, perform an in-tube DNase I treatment (off-column) using the protocol provided under the Appendix in the manual.