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
- HARVEST CELLS - Cells grown in a monolayer
For direct lysis in wells:- 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.
- Aspirate media of other all wells in plate in same manner. Change tip if you touch anything outside of the wells.
- Continue immediately with step two of the protocol.
- Add Buffer RLN to lyse plasma membrane (kept at 4°C)
For direct lysis of cells in cell-culture dishes (< 3.5 cm diameter)- Add 175μl cold Buffer RLN (4°C) to each well.
- Place plate on an ice-bucket and incubate for 5-7 min.
- Check under microscope after incubation. You should see many black dots, which are nuclei. This protocol only lyses cytoplasmic membrane.
- 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.
- Transfer to a microcentrifuge tube using p200 pipette and place tube on ice.
- Repeat this for all wells.
- Centrifuge lysate
- Centrifuge tube containing lysate at 4°C for 2 min at 300xg.
- Transfer supernatant to a new centrifuge tube and discard the pellet.
- Homogenize sample - Buffer RLT
Note: Ensure that β-ME is added to Buffer RLT before use.- Add 600μL Buffer RLT to clean tubes per each sample you have (have this ready before starting)
- 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.
- Mix thoroughly by vigorously vortexing.
- No further homogenization is required since genomic DNA is not released.
- 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 -/+ - Add Ethanol to adjust binding conditions
- Add 430 μl ethanol (100% Ethanol) to the homogenized lysate.
- Mix thoroughly by pipetting up and down. Do not centrifuge.
- Remove contaminants - Column and Buffers.
- Add 700ul of the sample, including any precipitate that may have formed, to an Rneasy mini column placed in a 2ml collection tube (supplied).
- Close the tube gently, and centrifuge for 30s at 11 krpm.
- 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.
- Repeat with the remaining amount of sample by adding rest of sample to same column, centrifuging and discarding flow-through.
- Reuse collection tube in step 7.
- DNase Treatment
- Pipette 350μl Buffer RW1 into the RNeasy mini column, and centrifuge for 30 s at 11 krpm (> 8000xg) to wash. Discard the flow-through.
- 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.
- 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.
- Pipette 350μl Buffer RW1 into the RNeasy mini column and centrifuge for 30s at 11 krpm. Discard the flow-through.
- Continue with the first Buffer RPE wash step in the relevant protocol.
- Buffer RPE - wash spins
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.- Transfer the RNeasy column into a new 2 ml collection tube (supplied).
- Pipet 500ul Buffer RPE onto the Rneasy column.
- Close gently, and centrifuge for 30sec at 11 krpm to wash the column.
- Discard the flow-though.
- Reuse the collection tube in step 9.
- Second wash with Buffer RPE
- Add another 500ul Buffer RPE to the RNeasy column.
- Close the tube gently, and centrifuge for 2 min at 11 krpm to dry the RNeasy silica-gel membrane.
- Ensure that there is no Buffer RPE carry-over
- 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.
- Centrifuge in a microcentrifuge at full speed for 1 min.
- ELUTE
- Transfer the RNeasy column to a new 1.5ml collection tube (supplied).
- Pipette 30-50ul RNase-free water directly onto the RNeasy silica-gel membrane (RNase-free water is supplied in the kit).
- Close the tube gently, and centrifuge for 1 min at 11 krpm to elute.
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.
Note: Do one plate at a time to prevent degradation of RNA.
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.
Note: You may stop at this step and place cells at -70°C. They should be stable for up to 6 months.
A precipitate may form after the addition of ethanol, but this will not affect the Rneasy procedure.
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.