RNA Extraction of Macrophages

RNA Extraction of Macrophages & On-Column DNase Treatment

QIAGEN RNeasy Mini Protocol for Isolation of Cytoplasm of Animal Cells
Spin Protocol

MATERIALS

  • RNeasy Mini Kit (QIAGEN Cat. No. 74104)
  • RLN Buffer, pre-cooled to 4°C, prepared as follows (final concentrations shown):
    • 50mM Tris-HCl, pH 8.0
    • 140mM NaCl
    • 1.5mM MgCl2
    • 0.5%(v/v) Nonidet P-40 (1.06 g/mL)
    • DEPC-treated H2O
    • Just before use, add: 1000U/ml Rnase inhibitor (optional), 1mM DTT (optional)
  • Buffer RLT
    • β-Mercaptoethanol (β-ME) must be added to Buffer RLT before use. β-ME is toxic; dispense in hood and wear protective clothing.
    • Add 10ul β-ME per 1mL of Buffer RLT.
    • Buffer RLT is stable for 1 month after addition of β-ME.
    • Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and then place at room temperature.
  • Buffer RPE is supplied as a concentrate:
    • Before using for the first time, add 4 volumes of ethanol (96% to 100%), to obtain a working solution, i.e., add 4x as much ethanol per volume of buffer
  • Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. Guanidine is an irritant. Take appropriate precautions and wear gloves when handling.
  • Cell lysis is performed on ice and following centrifugation at 4°C. All subsequent steps in the Rneasy protocol should be performed at room temperature. With the exception of buffer RLN, buffers should not be precooled. Obtain ice bucket for lysis reaction.
  • After cell harvesting and pelleting of nuclei, all centrifugation steps are performed at 20-25°C in a standard microcentrifuge. Ensure that the centrifuge does not cool below 20°C.
  • Pipettes: 5mL sterile pipettes; p20, p200 and p1000 pipetters and sterile tips: Spray with RNaseZap (Ambion Cat. No. 9780) and clean with 70% Ethanol before use.
  • Sterile microcentrifuge tubes.
  • Rubber cell-scraper (Fisher Cat. No. 08-773-2)
  • 100% Ethanol.
  • Cell cultures: this protocol is optimized for cell cultures in wells the size of 6-well plates, but can be adjusted for larger numbers of cells according to instructions in RNeasy protocol book.

Materials: DNase I treatment

  • RNase-Free DNase Set (QIAGEN Cat. No. 79254)
    • Prepare DNase I stock solution by dissolving the solid DNase I (1500 Kunitz units) in 550μl of Rnase-free water that is provided with the set.
    • Mix by gently inverting the tube, DO NOT VORTEX. The DNase is very sensitive to physical denaturation. Spin down before aliquoting for maximum recovery of protein.
    • Aliquot: For long term-storage, remove stock solution just prepared from the vial, and aliquot it into single-use aliquots, which can be
      store at -20°C for up to 9 months.
    • Thawed aliquots can be store at 2-8°C for up to 6 weeks. Do not refreeze aliquots after thawing.
    • Buffer RDD: Re-constituted DNase must be re-suspended in Buffer RDD before use as follows:
      • 10μl DNase/sample, and 10μl DNase:70μl RDD Buffer
      • Ex: 6 samples; 60μl DNase + 420μl RDD Buffer
    • Mix gently by inverting tube. Prepare and store at 4°C until ready to be added.
    • RNase-free water is provided with the kit.

PROTOCOL

  1. HARVEST CELLS - Cells grown in a monolayer
    For direct lysis in wells:
    1. Completely aspirate cell-culture medium of well 1 using a 5mL pipette. Tilt 6-well plate slightly to the side and submerge sterile pipette without touching monolayer. Make sure you aspirate all of the media, this is very important.
    2. Aspirate media of other all wells in plate in same manner. Change tip if you touch anything outside of the wells.
    3. Continue immediately with step two of the protocol.
  2. Note: Incomplete removal of the supernatant will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the Rneasy silica-gel membrane. Both effects may reduce RNA yield.

  3. Add Buffer RLN to lyse plasma membrane (kept at 4°C)
    For direct lysis of cells in cell-culture dishes (< 3.5 cm diameter)
    1. Add 175μl cold Buffer RLN (4°C) to each well.
    2. Place plate on an ice-bucket and incubate for 5-7 min.
    3. Check under microscope after incubation. You should see many black dots, which are nuclei. This protocol only lyses cytoplasmic membrane.
    4. Scrape plate gently using a rubber cell scraper. Not too harshly, as you may lyse nuclei and hurt the RNA. Scrape in single, circular motion.  Check under microscope that all cells have been removed.
    5. Transfer to a microcentrifuge tube using p200 pipette and place tube on ice.
    6. Repeat this for all wells.
  4. Note: Do one plate at a time to prevent degradation of RNA.

  5. Centrifuge lysate
    1. Centrifuge tube containing lysate at 4°C for 2 min at 300xg.
    2. Transfer supernatant to a new centrifuge tube and discard the pellet.
  6. The supernatant contains the cytoplasmic extract, and the RNA that is in it. It is generally slightly cloudy and yellow-white, depending on the cell type used. The pellet contains the nuclei and cell debris. The pellet is white and considerably smaller than the whole cell pellet obtained during harvesting.

  7. Homogenize sample - Buffer RLT
    Note: Ensure that β-ME is added to Buffer RLT before use.
    1. Add 600μL Buffer RLT to clean tubes per each sample you have (have this ready before starting)
    2. Transfer the supernatant containing cytoplasm of cells using a pipette (be careful not to touch the pellet) into a tube containing Buffer RLT. Do this for each tube.
    3. Mix thoroughly by vigorously vortexing.
    4. No further homogenization is required since genomic DNA is not released.
  8. Note: You may stop at this step and place cells at -70°C. They should be stable for up to 6 months.

  9. Record information for all samples
    Tube # Plate Condition (Concentration) Well # Genotype Notes
    1 1 Media (Ctrl) 1 +/+
    2 1 Media (Ctrl) 2 +/+
    3 1 Media (Ctrl) 3 +/+
    4 1 4 +/+
    5 1 5 +/+
    6 1 6 +/+
    7 2 1 -/+
    8 2 2 -/+
    9 2 3 -/+
    10 2 4 -/+
    11 2 5 -/+
    12 2 6 -/+
  10. Add Ethanol to adjust binding conditions
    1. Add 430 μl ethanol (100% Ethanol) to the homogenized lysate.
    2. Mix thoroughly by pipetting up and down. Do not centrifuge.
  11. A precipitate may form after the addition of ethanol, but this will not affect the Rneasy procedure.

  12. Remove contaminants - Column and Buffers.
    1. Add 700ul of the sample, including any precipitate that may have formed, to an Rneasy mini column placed in a 2ml collection tube (supplied).
    2. Close the tube gently, and centrifuge for 30s at 11 krpm.
    3. Discard the flow-through. Flow through is all that is collected on tube attached to column. Gently pull column out of tube and discard flow through by decanting into a beaker. Then place column back on tube, unless otherwise specified by protocol.
    4. Repeat with the remaining amount of sample by adding rest of sample to same column, centrifuging and discarding flow-through.
    5. Reuse collection tube in step 7.
  13. DNase Treatment
    1. Pipette 350μl Buffer RW1 into the RNeasy mini column, and centrifuge for 30 s at 11 krpm (> 8000xg) to wash. Discard the flow-through.
    2. Add 80μl DNase:REDD solution (10μl DNase to 70μl Buffer RDD per sample). Mix by gently inverting the tube. Note: DNase is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube; do not vortex. Prepare DNase and RDD mix before starting.
    3. Pipette the DNase I incubation mix (80μl) directly onto the RNeasy silica-gel membrane, and place on the bench for 15 min (20-30 °C). Note: Make sure to pipette the DNase incubation mix directly onto the RNeasy silica-gel membrane. Do this slowly so that mix does not splatter, adding directly on top of center of O-ring. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the RNeasy column.
    4. Pipette 350μl Buffer RW1 into the RNeasy mini column and centrifuge for 30s at 11 krpm. Discard the flow-through.
    5. Continue with the first Buffer RPE wash step in the relevant protocol.
  14. Buffer RPE - wash spins
    Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.
    1. Transfer the RNeasy column into a new 2 ml collection tube (supplied).
    2. Pipet 500ul Buffer RPE onto the Rneasy column.
    3. Close gently, and centrifuge for 30sec at 11 krpm to wash the column.
    4. Discard the flow-though.
    5. Reuse the collection tube in step 9.
  15. Second wash with Buffer RPE
    1. Add another 500ul Buffer RPE to the RNeasy column.
    2. Close the tube gently, and centrifuge for 2 min at 11 krpm to dry the RNeasy silica-gel membrane.
  16. It is important that the RNeasy silica-gel membrane is dry, since residual ethanol may interfere with downstream reactions. This centrifugation step ensures that no ethanol is carried over during elution.

  17. Ensure that there is no Buffer RPE carry-over
    1. Place the RNeasy column in a new 2ml collection tube (not supplied, can use a 1.5ml bullet tube), and discard the old collection tube with the flow-through. Remove column carefully so that the column does not contact the flow-through as this will result in carryover of ethanol.
    2. Centrifuge in a microcentrifuge at full speed for 1 min.
  18. ELUTE
    1. Transfer the RNeasy column to a new 1.5ml collection tube (supplied).
    2. Pipette 30-50ul RNase-free water directly onto the RNeasy silica-gel membrane (RNase-free water is supplied in the kit).
    3. Close the tube gently, and centrifuge for 1 min at 11 krpm to elute.