Protocol for Total RNA Isolation from Cells

  • Document # 27175
  • Version 1.0.0
  • Dec 2019

The following protocol is for total RNA isolation from cells using the Total RNA Purification Kit (Catalog #79040). For complete instructions, refer to the Technical Manual (Document #10000005434).

Directions

A. Preparation of Cell Lysate

Prepare cell lysate from adherent cells or cells in suspension, as indicated below.


From Adherent Cells

  1. Aspirate cell culture medium. Add ice-cold sterile D-PBS as indicated in Table 1. Aspirate D-PBS.

  2. Table 1. Recommended Volumes of PBS, RNA Lysis Buffer + TG, and 100% Isopropanol for Various Cultureware

    Cultureware
    Volume of D-PBS
    Volume of RNA Lysis Buffer + TG
    Volume of 100% Isopropanol
    T-25 cm2 flask
    5 mL
    500 µL
    170 µL
    6-well plate
    2 mL
    250 µL
    85 µL
    24-well plate
    500 µL
    100 µL
    35 µL
    48-well plate
    250 µL
    100 µL
    35 µL
    96-well plate
    100 µL
    100 µL
    35 µL

  3. Add RNA Lysis Buffer + TG as indicated in Table 1. Gently rock the plate or flask to completely cover the adherent cells with buffer. Pipette the lysate up and down over the cultureware surface 7 – 10 times.
  4. Collect the lysate and transfer to a new microcentrifuge tube.
  5. Add 100% isopropanol as indicated in Table 1. Mix by vortexing for 5 seconds.
  6. Proceed to RNA isolation.

From Cells in Suspension

  1. In a sterile centrifuge tube, centrifuge cell suspension at 300 x g for 5 minutes. Remove and discard supernatant.
  2. Add ice-cold, sterile D-PBS to wash cells. Centrifuge at 300 x g for 5 minutes. Remove as much supernatant as possible and discard.
  3. Add RNA Lysis Buffer + TG as indicated in Table 2. Mix well by pipetting up and down 7 - 10 times, or by vortexing. For > 2 x 106 cells, pass the lysate through a 20-gauge needle 4 - 5 times to shear the genomic DNA.
  4. Add 100% isopropanol as indicated in Table 2. Mix by vortexing for 5 seconds.

  5. Table 2. Recommended Volumes of RNA Lysis Buffer + TG and Isopropanol per Cell Input Range

    Cell Input Range
    Volume of RNA Lysis Buffer + TG
    Volume of 100% Isopropanol
    1 x 102 to 5 x 105
    100 µL
    35 µL
    > 5 x 105 to 2 x 106
    250 µL
    85 µL
    > 2 x 106 to 5 x 106
    500 µL
    170 µL

  6. Proceed to RNA isolation.

B. RNA Isolation

  1. Insert minicolumn into Collection Tube.
  2. Transfer the lysate to the minicolumn assembly.
  3. Centrifuge at 12,000 - 14,000 x g for 30 seconds. Discard the liquid in the Collection Tube and reinsert minicolumn into Collection Tube.
  4. Add 500 μL RNA Wash Buffer to the minicolumn. Centrifuge at 12,000 - 14,000 x g for 30 seconds. Discard the liquid in the Collection Tube and reinsert minicolumn into Collection Tube.
  5. Prepare DNase I Incubation Mix by combining the reagents as indicated in Table 3, per sample, in the order listed.

  6. Table 3. Preparation of DNase I Incubation Mix

    Component
    Volume per Sample
    DNA Digestion Buffer
    24 µL
    MnCl2
    3 µL
    DNase I Solution
    3 µL

  7. Mix by gently pipetting up and down; do not vortex. Store on ice.
  8. Add 30 μL of fresh DNase I Incubation Mix directly to the minicolumn membrane. Incubate at room temperature (15 - 25°C) for 15 minutes.
  9. Add 200 μL of Column Wash Buffer (with ethanol added) to the minicolumn. Centrifuge at 12,000 - 14,000 x g for 15 seconds.
  10. Add 500 μL of RNA Wash Buffer (with ethanol added) to the minicolumn. Centrifuge at 12,000 - 14,000 x g for 30 seconds. Remove the minicolumn and transfer to a new Collection Tube. Discard the Collection Tube containing wash buffer.
  11. Add 300 μL of RNA Wash Buffer to the minicolumn. Centrifuge at high speed for 2 minutes.
  12. Transfer minicolumn to an Elution Tube. Add nuclease-free water to the minicolumn membrane as indicated in Table 4. Incubate at room temperature for 1 - 2 minutes. Centrifuge at 12,000 - 14,000 x g for 1 minute.

  13. Table 4. Recommended Volume of Nuclease-Free Water per Input Cell Range

    Cell Input Range
    Volume of Nuclease-Free Water
    1 x 102 to 5 x 105
    15 µL
    > 5 x 105 to 2 x 106
    30 µL
    > 2 x 106 to 5 x 105
    50 µL

  14. Remove and discard minicolumn. Store purified RNA at -80ºC.