Guidelines for Purification of RNA from Cultured Mammalian Cells

  1. RNA can be purified from as few as 100 cells up to as many as 1x107 cells in a standard prep.

    Total RNA was isolated from varying amounts of HeLa cells, 100 cells up to 1x106 cells. Samples were analyzed on a Bioanalyzer® Pico chip, with RIN values and total yields shown below the lanes (A). Electropherograms are included as a reference (B).


  2. Monarch DNA/RNA Protection Reagent is not required for this protocol. Cells are lysed in the RNA Lysis Buffer.

  3. For cells stabilized in DNA/RNA Protection Reagent, allow samples to equilibrate to room temperature prior to the addition of an equal volume of RNA Lysis Buffer.

  4. If working with frozen cell pellets, thaw briefly (30-40 seconds at room temperature) prior to resuspension in RNA Lysis Buffer.

  5. RNA Lysis Buffer volume recommendations are for pelleted cells, however, cells in suspension can be directly processed by adding 4 volumes of RNA Lysis Buffer.

  6. After lysis, do not place samples on ice. Keep samples at room temperature to prevent precipitation of detergent in the lysis buffer. All subsequent steps should be performed at room temperature.

  7. Proteinase K digestion is not required for processing cultured mammalian cells.

 

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