General Guidelines for Successful RNA Purification Using the
Monarch Total RNA Miniprep Kit
Before your Prep:
- 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.
- Ensure that you are using the correct protocol and ensure that you have all necessary materials and equipment on-hand. The protocol consists of two parts: Part 1: Sample Disruption 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.
- If you are working with tissue or leukocytes, 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. When working with mammalian whole blood the Proteinase K incubation is performed at room temperature.
- Fresh samples should be rapidly processed after harvest, flash frozen for later use, or stored in a stabilization reagent such as Monarch DNA/RNA Protection Reagent (included in the kit). See Monarch DNA/RNA Protection Reagent Guidance in the product manual.
- Keep frozen samples frozen until mixed with Monarch DNA/RNA Protection Reagent. One exception is the processing of frozen mammalian cell culture pellets. We recommend briefly thawing pellets at room temperature before adding RNA Lysis Buffer (see product manual for details).
- Choose input amounts carefully to ensure buffer volumes are adequate and columns are not overloaded. Improper input amounts can reduce yield, purity, and integrity of the RNA.
- Determine whether you will need 1X or 2X Monarch DNA/RNA Protection Reagent. Different sample types require different concentrations of Protection Reagent, so it is important to follow the protocol and to dilute only the volume that you need. This reagent is not necessary if working with cultured mammalian cells.
- Ensure your samples are completely disrupted/homogenized to release RNA and maximize yield. For tissue samples, mechanical homogenization prior to digestion with Proteinase K often increases RNA yield. When working with tough-to-lyse samples (e.g., bacteria, yeast, plant etc), we recommend mechanical homogenization with a bead homogenizer. If multiple rounds of homogenization are recommended, place samples on ice for one minute between rounds to prevent over-heating.
- Do not place samples on ice after adding RNA Lysis Buffer. Addition of RNA Lysis Buffer and all subsequent steps should be performed at room temperature to prevent precipitation of detergents in buffers.
- Label the collection tube fitted to the gDNA Removal Column and SAVE THE FLOW-THROUGH for this step. Since the gDNA Purification Column will be discarded after spinning, it is important to label the collection tube for sample identification. After spinning the gDNA Removal Column, SAVE THE FLOW-THROUGH as RNA partitions into the flow-through. Addition of ethanol to the flow-through creates favorable conditions for RNA to bind to the RNA Purification Column.
- If 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 RNA Wash buffer step prior to adding the enzyme. Also, it can help to prepare a master mix of DNase I and DNase I Reaction Buffer if you will perform multiple preps at one time.
- Perform all wash steps. The washes will remove contaminants, including DNase I and salts; it is particularly important to spin the RNA Purification Column for 2 minutes after the last wash.
- Don’t let the tip of the RNA Purification Column contact the flow-through in the collection tube after the final wash to prevent salt and/or ethanol carry-over. If unsure, repeat centrifugation.
- Adjust your elution volume depending on your downstream needs. The standard elution volume is 100 µl, however RNA can be eluted with 50 µl if you require a more concentrated solution or a smaller volume.
- Place eluted RNA on ice for immediate use or freeze for long-term storage. Additionally, it is good practice to store eluted RNA in aliquots to avoid excessive freeze-thaw cycles.
- Spin your sample before taking an aliquot for spectrophotometric analysis. On occasion, silica particles may be found in the eluate. To ensure that this will not affect the OD260/230 ratio, spin the eluate for 1-2 minutes at 16,000 x g, and then remove an aliquot from the top of the liquid.
- Add EDTA to the eluted RNA (to 0.1-1 mM) if it will be stored for an extended period of time. Adding EDTA may protect RNA samples that will be stored for an extended time.
- If further gDNA removal is essential for downstream applications, perform an in-tube DNase I treatment( off-column). The protocol can be found online or in the Supplemental Protocols section of the product manual.
- Large (>200 nt) and small (<200 nt) RNAs can be fractionated according to size after purification. Refer to the product manual for instructions.