Guidelines for RNA Purification from Tissues

  1. Use an appropriate amount of starting material. This ensures sufficient RNA yield without exceeding the binding capacity of the column. Too much sample may reduce lysis efficiency, introduce excessive amounts of cellular components other than RNA, and compromise RNA binding to the RNA Purification Column. It is important to note that the maximum amount of starting material varies for low yield (e.g., brain and muscle) and high yield (e.g., spleen and liver) tissues.

  2. Fresh or frozen tissue can be processed using the Monarch Total RNA Miniprep Kit (NEB #T2010). In general, samples should be flash frozen or rapidly processed after harvest to ensure RNA remains intact and accurately reflects the gene expression profile at the time of harvest. Process fresh or frozen samples as follows:

    1. Fresh tissue (up to 20 mg): After harvesting, keep tissue on ice and process immediately by mixing sample with Monarch DNA/RNA Protection Reagent (NEB #T2011). Endogenous nucleases can quickly degrade RNA and negatively impact results.

    2. Frozen tissue (up to 20 mg) not previously stored in stabilization reagent: Keep tissue frozen until mixed with Monarch DNA/RNA Protection Reagent (NEB #T2011). 

    3. Fresh or frozen tissue pieces greater than 20 mg: Immediately homogenize samples and/or digest with Proteinase K after addition of Monarch DNA/RNA Protection Reagent. If necessary, place samples mixed with Protection Reagent on ice briefly until they can be processed.

    4. Tissues stored in Monarch DNA/RNA Protection Reagent: Allow samples to equilibrate to room temperature.  Ensure the volume of Protection Reagent is consistent with protocol recommendations.

    5. Tissue stored in RNAlater®: Remove RNAlater prior to adding DNA/RNA Protection Reagent.

  3. Be sure to use the Monarch DNA/RNA Protection Reagent at a 1X concentration. Dilute the appropriate volume of the supplied 2X Protection Reagent concentrate with nuclease-free water depending on the number of samples you are processing.

  4. Mechanical homogenization prior to digestion with Proteinase K often increases RNA yield.

  5. Doubling the amount of Proteinase K and/or increasing incubation time may also increase yield for some tissues.

  6. Addition of Monarch RNA Lysis Buffer (NEB #T2012) and all subsequent steps should be carried out at room temperature.

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